Phylogeny and systematics of the genus Calonectria.

Species of Calonectria are important plant pathogens, several of which have a worldwide distribution. Contemporary taxonomic studies on these fungi have chiefly relied on DNA sequence comparisons of the beta-tubulin gene region. Despite many new species being described, there has been no phylogenetic synthesis for the group since the last monographic study almost a decade ago. In the present study, the identity of a large collection of Calonectria isolates from various geographic regions was determined using morphological and DNA sequence comparisons. This resulted in the discovery of seven new species; Ca. densa, Ca. eucalypti, Ca. humicola, Ca. orientalis, Ca. pini, Ca. pseudoscoparia and Ca. sulawesiensis, bringing the total number of currently accepted Calonectria species to 68. A multigene phylogeny was subsequently constructed for all available Calonectria spp., employing seven gene regions, namely actin, beta-tubulin, calmodulin, histone H3, the internal transcribed spacer regions 1 and 2 and the 5.8S gene of the ribosomal RNA, 28S large subunit RNA gene and translation elongation 1-alpha. Based on these data 13 phylogenetic groups could be distinguished within the genus Calonectria that correlated with morphological features. Dichotomous and synoptic keys to all Calonectria spp. currently recognised are also provided.

INTRODUCTION

The genus Calonectria (Ca.) was first described in 1867, with Ca. daldiniana as the type. This species was later reduced to synonymy with Ca. pyrochroa based on morphological comparisons by Rossman (1979). Calonectria spp. are Euascomycetes in the order Hypocreales (Hibbett , Schoch ) and are characterised by their yellow to dark red perithecia, with scaly to warty ascocarp walls giving rise to long-stalked, clavate asci with 1–multi-septate ascospores and Cylindrocladium (Cy.) anamorphs (Rossman 1993, Crous 2002, Lombard ). The genus Cylindrocladium was described by Morgan (1892), and is characterised by branched conidiophores with stipe extensions terminating in characteristic vesicles and producing cylindrical, 1–multi-septate conidia (Crous & Wingfield 1994, Crous 2002). Morphologically, the anamorph provides the greatest number of distinguishing characters for Calonectria and it is also the state most frequently encountered in nature (Peerally 1991, Crous & Wingfield 1994, Schoch , Crous 2002). Consequently, species of Calonectria are primarily distinguished by their anamorph characters, especially vesicle shape, stipe extension length, conidial septation, and dimensions on a standardised medium under defined growth conditions (Boesewinkel 1982, Peerally 1991, Crous & Wingfield 1994, Crous 2002). Despite, the use of standardised conditions, taxonomic confusion can result because some intraspecific variation in vesicle shape and conidial dimension is common (Crous & Peerally 1996, Crous ).

The reliability of vesicle shape as a distinguishing morphological character has been questioned (Sober & Alfieri 1972, Hunter & Barnett 1978, Rossman 1983), although Crous et al. (1992) demonstrated experimentally that the shape of this structure can be influenced by the osmotic potential of the medium and the age of the culture, but that it remains a reliable morphological feature if these aspects are standardised. In the original description of Ca. morganii (= Cy. scoparium), the type of the anamorph, Morgan (1892) failed to include details of the stipe extension and terminal vesicle, which is a defining characteristic in distinguishing anamorphs of Calonectria (Boesewinkel 1982, Peerally 1991, Crous & Wingfield 1994, Crous 2002).

Calonectria spp. produce three different morphological forms of conidia, of which the macroconidia are present in all but Ca. multiseptata (Peerally 1991, Crous & Wingfield 1994, Crous et al. 1998b, Crous 2002). Mega- and microcondia are less frequently encountered and these are not regarded as important characters to distinguish between species (Sober 1971, Crous & Wingfield 1994, Crous & Seifert 1998, Crous 2002). Similar to vesicle shape, significant variability can occur in the production of all conidial types, so that this feature alone is not always a reliable taxonomic character to define species.

Both homothallic and heterothallic mating systems are found amongst species of Calonectria (Alfieri , Schubert , Crous & Wingfield 1994, Crous 2002). Heterothallic Calonectria spp. have a biallelic heterothallic mating system with the female structures (protoperithecia) spermatised by conidia or hyphae of an opposite mating type strain (Schoch et al. 1999, 2000a, 2001a). Some Calonectria spp. have retained the ability to recombine with other closely related Calonectria spp., although the progeny from these crosses have low levels of fertility (Crous 2002). This has complicated the application of the biological species concept for Calonectria, although it has been useful for some species (Schoch , Lombard ).

Difficulties experienced in morphological identification, have led to several molecular approaches being employed to identify Calonectria spp. These include total protein electrophoresis (Crous , El-Gholl ), isozyme electrophoresis (El-Gholl , El-Gholl , Crous ), random amplification of polymorphic DNA (RAPD) (Overmeyer , Victor , Schoch , Risède & Simoneau 2004), restriction fragment length polymorphisms (RFLP) (Crous , Crous , Crous , Jeng , Victor , Risède & Simoneau 2001) and DNA hybridisation (Crous et al. 1993a, 1995, 1997, Victor ). However, DNA sequence comparisons and associated phylogenetic inference has had the most significant impact on the taxonomy of the group. It is also most widely applied in contemporary species descriptions. The 5.8S ribosomal RNA gene and flanking internally transcribed spacer (ITS) sequences made it possible for Jeng et al. (1997) to distinguish between Cy. scoparium and Cy. floridanum isolates. Subsequently, it was found that this gene region contains few informative characters for members of the genus (Crous , Schoch , Risède & Simoneau 2001, Schoch ). As a consequence, this resulted in the β-tubulin (BT) (Schoch ) and histone H3 (HIS3) (Kang ) gene regions being widely employed to improve the resolution of phylogenetic trees for species of Calonectria.

The first complete DNA sequence-based phylogenetic study using partial BT gene sequences (Schoch ) compared phenotypic, biological and phylogenetic species concepts used in the taxonomy of Calonectria. Results showed that the genus represents a well resolved monophyletic lineage. Subsequently, combined DNA sequence data for the ITS, BT and HIS3 gene regions have been used to resolve taxonomic questions for Calonectria (Schoch , Henricot & Culham 2002, Crous et al. 2004b, 2006). Other DNA sequences recently used to distinguish between species include the translation elongation factor 1–alpha (TEF-1α) and calmodulin (CAL) gene regions (Crous , Lombard et al. 2009, 2010a, b). However, sequence data for these regions on GenBank (www.ncbi.nlm.nih.gov) are incomplete for the group, substantially reducing their value.

The aim of this study was to consider the identity of a large collection of previously unidentified Calonectria isolates collected over a five year period from various parts of the world. Morphological characteristics, phylogenetic inference and mating compatibility were employed for this purpose. Subsequently, the phylogenetic relationships between Calonectria spp. were re-evaluated by constructing a multigene phylogeny for seven gene regions and considering these results together with morphological features for all species in the genus.

MATERIALS AND METHODS

Isolates

Plant material showing symptoms of Calonectria infections as well as soil samples were collected from various geographical regions over a period of five years. Diseased plant material was placed in moist chambers and incubated for 48 h at room temperature to induce sporulation. Direct isolations were made onto malt extract agar (2 % w/v; MEA; Biolab, Midrand, South Africa) and cultures were incubated for 7 d at 25 °C under continuous near-ultraviolet light. Baiting, using seeds of Medicago sativa, was applied for the soil samples following the technique of Crous (2002). For each isolate, single conidial cultures were prepared on MEA. Representative strains are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa and the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands (Table 1).

Table 1.

Isolates of Calonectria spp. studied.

Species	Isolate number1	Other collections1	GenBank accession nr.2							Reference3
			ACT	BT	CAL	HIS3	ITS	LSU	TEF-1α
Ca. acicola	CBS 114812		GQ280424	DQ190590	GQ267359	DQ190692	GQ280546	GQ280668	GQ267291	Gadgil & Dick (2004)
	CBS 114813T	CMW 30996	GQ280425	DQ190591	GQ267360	DQ190693	GQ280547	GQ280669	GQ267292
Ca. angustata	CBS 109065T	CMW 30990 = CPC 2347 = P99-0454	GQ280426	AF207543	GQ267361	DQ190696	GQ280548	GQ280671	FJ918551	Crous (2002)
	CBS 109169	CMW 30983 = CPC 3152 = P99-1321	GQ280427	DQ190593	GQ267362	DQ190695	GQ280549	GQ280670	FJ918552
Ca. asiatica	CBS 112711	CPC 3898 = SFE 744	GQ280429	AY725613	AY725738	AY725655	GQ280551	GQ280673	AY725702	Crous et al. (2004b)
	CBS 114073T	CMW 23782 = CPC 3900 = SFE 726	GQ280428	AY725616	AY725741	AY725658	GQ280550	GQ280672	AY725705
Ca. australiensis	CBS 112954T	CMW 23669 = CPC 4714	GQ280430	DQ190596	GQ267363	DQ190699	GQ280552	GQ280674	GQ267293	Crous et al. (2006)
Ca. avesiculata	CBS 313.92T	CMW 23670 = CPC 2373 = ATCC 38226	GQ280431	AF333392	GQ267364	DQ190620	GQ280553	GQ280675	GQ267294	Crous (2002)
Ca. brachiatica	CBS 123700T	CMW 25298	GQ280433	FJ696388	GQ267366	FJ696396	GQ280555	GQ280677	GQ267296	Lombard et al. (2009)
	CMW 25302		GQ280432	FJ716708	GQ267365	FJ716712	GQ280554	GQ280676	GQ267295
Ca. brassicae	CBS 111478	CMW 30981	GQ280455	DQ190611	GQ267383	DQ190719	GQ280577	GQ280699	FJ918567	Crous (2002)
	CBS 111869T	CMW 30982 = CPC 2409 = PC 551197	GQ280454	AF232857	GQ267382	DQ190720	GQ280576	GQ280698	FJ918566
Ca. brasiliensis	CBS 230.51T	CMW 23670 = CPC 2390	GQ280502	GQ267241	GQ267421	GQ267259	GQ280624	GQ280746	GQ267328	Lombard et al. (2009c)
	CBS 114257	CMW 32949 = CPC 1944	GQ280503	GQ267242	GQ267422	GQ267260	GQ280625	GQ280747	GQ267329
Ca. canadensis	CBS 110817T	CMW 23673 = CPC 499	GQ280434	AF348212	AY725743	AF348228	GQ280556	GQ280678	GQ267297	Crous (2002)
Ca. cerciana	CBS 123693T	CMW 25309	GQ280437	FJ918510	GQ267369	FJ918528	GQ280559	GQ280681	FJ918559	Lombard et al. (2010c)
	CBS 123695	CMW 25290	GQ280438	FJ918511	GQ267370	FJ918529	GQ280560	GQ280682	FJ918560
Ca. chinensis	CBS 112744	CMW 30986 = CPC 4104	GQ280440	AY725618	AY725746	AY725660	GQ280562	GQ280684	AY725709	Crous et al. (2004b)
	CBS 114827T	CMW 23674 = CPC 4101	GQ280390	AY725619	AY725747	AY725661	GQ280561	GQ280683	AY725710
Ca. citri	CBS 186.36T	CMW 23675	GQ280441	AF333393	GQ267371	GQ267247	GQ280563	GQ280685	GQ267299	Crous (2002)
Ca. clavata	CBS 114557T	CMW 23690 = CPC 2536 = ATCC 66389	GQ280449	AF333396	GQ267377	DQ190623	GQ280571	GQ280693	GQ267305	Crous (2002)
	CBS 114666T	CMW 30994 = CPC 2537	GQ280450	DQ190549	GQ267378	DQ190624	GQ280572	GQ280694	GQ267306
Ca. colhounii	CBS 293.79T	CMW 30999	GQ280443	DQ190564	GQ267373	DQ190639	GQ280565	GQ280687	GQ267301	Crous (2002)
	CBS 114704		GQ280442	DQ190563	GQ267372	DQ190638	GQ280564	GQ280686	GQ267300
Ca. colombiana	CBS 115127T	CMW 30871 = CPC 1160	GQ280538	FJ972423	GQ267455	FJ972442	GQ280660	GQ280782	FJ972492	Lombard et al. (2010a)
	CBS 115638	CMW 30766 = CPC 1161	GQ280539	FJ972422	GQ267456	FJ972441	GQ280661	GQ280783	FJ972491
Ca. colombiensis	CBS 112220T	CMW 23676 = CPC 723	GQ280444	GQ267207	AY725748	AY725662	GQ280566	GQ280688	AY725711	Crous et al. (2004b)
	CBS 112221	CMW 30985 = CPC 724	GQ280445	AY725620	AY725749	AY725663	GQ280567	GQ280689	AY725712	Crous (2002)
Ca. curvispora	CBS 116159T	CMW 23693	GQ280446	AF333394	GQ267374	AY725664	GQ280568	GQ280690	GQ267302	Crous (2002)
Ca. densa	CBS 125249	CMW 31184	GQ280523	GQ267230	GQ267442	GQ267279	GQ280645	GQ280767	GQ267350	This study
	CBS 125250	CMW 31185	GQ280524	GQ267231	GQ267443	GQ267280	GQ280646	GQ280768	GQ267351
	CBS 125261T	CMW 31182	GQ280525	GQ267232	GQ267444	GQ267281	GQ280647	GQ280769	GQ267352
Ca. ecuadoriae	CBS 111394	CMW 30980 = CPC 1628	GQ280448	DQ190599	GQ267376	DQ190704	GQ280570	GQ280692	GQ267304	Crous et al. (2006)
	CBS 111406T	CMW 23677 = CPC 1635	GQ280447	DQ190600	GQ267375	DQ190705	GQ280569	GQ280691	GQ267303
Ca. eucalypti	CBS 125273	CMW 14890	GQ280510	GQ267217	GQ267429	GQ267266	GQ280632	GQ280754	GQ267337	This study
	CBS 125274	CMW 18443	GQ280509	GQ267216	GQ267428	GQ267265	GQ280631	GQ280753	GQ267336
	CBS 125275T	CMW18444	GQ280511	GQ267218	GQ267430	GQ267267	GQ280633	GQ280755	GQ267338
	CBS 125276	CMW 18445	GQ280512	GQ267219	GQ267431	GQ267268	GQ280634	GQ280756	GQ267339
Ca. gordoniae	CBS 112142	CMW 23694 = CPC 3136 = ATCC 201837	GQ280453	AF449449	GQ267381	DQ190708	GQ280575	GQ280697	GQ267309	Leahy et al. (2000)
Ca. gracilipes	CBS 111141T		GQ280457	DQ190566	GQ267385	DQ190644	GQ280579	GQ280701	GQ267311	Crous (2002)
	CBS 115674		GQ280456	AF333406	GQ267384	DQ190645	GQ280578	GQ280700	GQ267310
Ca. gracilis	CBS 111284		GQ280489	DQ190567	GQ267408	DQ190647	GQ280611	GQ280733	GQ267324	Crous (2002)
	CBS 111807		GQ280488	AF232858	GQ267407	DQ190646	GQ280610	GQ280734	GQ267323
Ca. hawksworthii	CBS 111870T	CPC 2405 = MUCL 30866	GQ280458	AF333407	GQ267386	DQ190649	GQ280580	GQ280702	FJ918558	Crous (2002)
Ca. hongkongensis	CBS 114711	CMW 30995	GQ280460	AY725621	AY725754	AY725666	GQ280582	GQ280704	AY725716	Crous et al. (2004b)
	CBS 114828T		GQ280459	AY725622	AY725755	AY725667	GQ280581	GQ280703	AY725717
Ca. humicola	CBS 125251T	CMW 31183	GQ280526	GQ267233	GQ267445	GQ267282	GQ280648	GQ280770	GQ267353	This study
	CBS 125252	CMW 31186	GQ280527	GQ267234	GQ267446	GQ267283	GQ280649	GQ280771	GQ267354
	CBS 125269	CMW31187	GQ280528	GQ267235	GQ267447	GQ267284	GQ280650	GQ280772	GQ267355
Ca. hurae	CBS 114551	CMW 16720 = CPC 2344	GQ280461	AF333408	GQ267387	DQ190728	GQ280583	GQ280705	FJ918548	Crous (2002)
Ca. ilicicola	CBS 190.50T	CMW 30998 = CPC 2482 = IMI 299389	GQ280483	AY725631	AY725764	AY725676	GQ280605	GQ280727	AY725726	Crous (2002)
	CBS 115897		GQ280484	AY725647	GQ267403	GQ267256	GQ280606	GQ280728	AY725729
Ca. indonesiae	CBS 112823T	CMW 23683 = CPC 4508	GQ280463	AY725623	AY725756	AY725668	GQ280585	GQ280707	AY725718	Crous et al. (2004b)
	CBS 112840	CPC 4547	GQ280464	AY725625	AY725758	AY725670	GQ280586	GQ280708	AY725720
Ca. indusiata	CBS 144.36	CMW 23699	GQ280536	GQ267239	GQ267453	GQ267262	GQ280658	GQ280780	GQ267332	Crous (2002)
	CBS 114684	CPC 2446 = UFV 16A	GQ280537	AF232862	GQ267454	DQ190652	GQ280659	GQ280781	GQ267333
Ca. insularis	CBS 114558T	CMW 30991	GQ280465	AF210861	GQ267389	FJ918526	GQ280587	GQ280709	FJ918556	Crous (2002)
	CBS 114559	CMW 30992	GQ280466	AF210862	GQ267390	FJ918525	GQ280588	GQ280710	FJ918555
Ca. kyotensis	CBS 170.77	CMW 23679 = IMI 299388	GQ280452	GQ267209	GQ267380	GQ267249	GQ280574	GQ280696	GQ267308	Crous (2002)
	CBS 413.67	CMW 23678 = CPC 2391	GQ280451	GQ267208	GQ267379	GQ267248	GQ280573	GQ280695	GQ267307
Ca. leguminum	CBS 728.68T	CMW 23684 = IMI 299578	GQ280467	AF389837	GQ267391	DQ190654	GQ280589	GQ280711	FJ918547	Crous (2002)
Ca. leucothoës	CBS 109166	CMW 30977 = CPC 3612 = P97-2605	GQ280468	FJ918508	GQ267392	FJ918523	GQ280590	GQ280712	FJ918553	Crous (2002)
Ca. macroconidialis	CBS 114880T	CPC 307	GQ280469	AF232855	GQ267393	DQ190655	GQ280591	GQ280713	GQ267313	Crous (2002)
Ca. madagascariensis	CBS 114571	CMW 30993 = CPC 2253	GQ280471	DQ190571	GQ267395	DQ190657	GQ280593	GQ280715	GQ267315	Crous (2002)
	CBS 114572T	CMW 23686 = CPC 2252	GQ280470	DQ190572	GQ267394	DQ190658	GQ280592	GQ280714	GQ267314
Ca. malesiana	CBS 112710	CPC 3899	GQ280473	AY725626	AY725759	AY725671	GQ280595	GQ280717	AY725721	Crous et al. (2004b)
	CBS 112752T	CMW 23687 = CPC 4223	GQ280472	AY725627	AY725760	AY725672	GQ280594	GQ280716	AY725722
Ca. mexicana	CBS 110918T	CMW 9055	GQ280474	AF210863	GQ267396	FJ972460	GQ280596	GQ280718	FJ972526	Crous (2002)
Ca. morganii	CBS 110666	CMW 30978 = P90.1479	GQ280504	FJ918509	GQ267423	FJ918527	GQ280626	GQ280748	FJ9188557	Crous (2002)
Ca. multiphialidica	CBS 112678	CMW 23688	GQ280475	AY725628	AY725761	AY725673	GQ280597	GQ280719	AY725723	Crous et al. (2004b)
Ca. multiseptata	CBS 112682	CMW 23692 = CPC 1589	GQ280476	DQ190573	GQ267397	DQ190659	GQ280598	GQ280720	FJ918535	Crous (2002)
Ca. naviculata	CBS 101121T	CMW 30974	GQ280478	GQ267211	GQ267399	GQ267252	GQ280600	GQ280722	GQ267317	Crous (2002)
	CBS 116080	CMW 16723	GQ280477	AF333409	GQ267398	GQ267251	GQ280599	GQ280721	GQ267316
Ca. orientalis	CBS 125258	CMW 20272	GQ280531	GQ267238	GQ267450	GQ267287	GQ280653	GQ280775	GQ267358	This study
	CBS 125259	CMW 20273	GQ280530	GQ267237	GQ267449	GQ267286	GQ280652	GQ280774	GQ267357
	CBS 125260T	CMW 20291	GQ280529	GQ267236	GQ267448	GQ267285	GQ267651	GQ280773	GQ267356
Ca. ovata	CBS 111299	CMW 16724	GQ280479	GQ267212	GQ267400	GQ267253	GQ280601	GQ280723	GQ267318	Crous (2002)
	CBS111307	CMW 30979	GQ280480	AF210868	GQ267401	GQ267254	GQ280602	GQ280724	GQ267319
Ca. pacifica	CBS 109063	CMW 16726 = IMI 35428	GQ280481	GQ267213	AY725762	GQ267255	GQ280603	GQ280725	AY725724	Crous (2002)
	CBS 114038	CMW 30988	GQ280482	AY725630	GQ267402	AY725675	GQ280604	GQ280726	GQ267320
Ca. pauciramosa	CMW 5683T	CPC 971	GQ280486	FJ918514	GQ267405	FJ918531	GQ280608	GQ280730	FJ918565	Crous (2002)
	CMW30823	CPC 416	GQ280485	FJ918515	GQ280404	FJ918532	GQ280607	GQ280729	FJ918566
Ca. penicilloides	CBS 174.55T	CMW 23696	GQ280487	AF333414	GQ267406	GQ267257	GQ280609	GQ280731	GQ267322	Crous (2002)
Ca. pini	CBS 123698T	CMW 31209	GQ280517	GQ267224	GQ267436	GQ267273	GQ280639	GQ280761	GQ267344	This study
	CBS 125523	CMW 31210	GQ280518	GQ267225	GQ267437	GQ267274	GQ280640	GQ280762	GQ267345
Ca. polizzii	CBS 125270	CMW 7804	GQ280544	FJ972417	GQ267461	FJ972436	GQ280666	GQ280788	FJ972486	Lombard et al. (2010a)
	CBS 125271	CMW 10151	GQ280545	FJ972418	GQ267462	FJ972437	GQ280667	GQ280789	FJ972487
Ca. pseudonaviculata	CBS 114417T	CMW 23672	GQ280490	GQ267214	GQ267409	GQ267258	GQ280612	GQ280734	GQ267325	Crous et al. (2002)
Ca. pseudoreteaudii	CBS 123694T	CMW 25310	GQ280492	FJ918504	GQ267411	FJ918519	GQ280614	GQ280736	FJ918541	Lombard et al. (2010c)
	CBS 123696	CMW 25292	GQ280491	FJ918505	GQ267410	FJ918520	GQ280613	GQ280735	FJ918542
Ca. pseudoscoparia	CBS 125254	CMW 15214	GQ280519	GQ267226	GQ267438	GQ267275	GQ280641	GQ280763	GQ267346	This study
	CBS 125255	CMW 15215	GQ280520	GQ267227	GQ267439	GQ267276	GQ280642	GQ280764	GQ267347
	CBS 125256	CMW 15216	GQ280521	GQ267228	GQ267440	GQ267277	GQ280643	GQ280765	GQ267348
	CBS 125257T	CMW 15218	GQ280522	GQ267229	GQ267441	GQ267278	GQ280644	GQ280766	GQ267349
Ca. pseudospathiphylli	CBS 109162T	CMW 30976 = CPC 1623	GQ280493	FJ918513	GQ267412	AF348241	GQ280615	GQ280737	FJ918562	Crous (2002)
Ca. pteridis	CBS 111793T	CMW 16736 = CPC 2372 = ATCC 34395	GQ280494	DQ190578	GQ267413	DQ190679	GQ280616	GQ280738	FJ918563	Crous (2002)
	CBS 111871	CMW 30982 = CPC 2443	GQ280495	DQ190579	GQ267414	DQ190680	GQ280617	GQ280739	FJ918564
Ca. pyrochoa	CBS 749.70T	CMW 23682	GQ280462	GQ267210	GQ267388	GQ267250	GQ280584	GQ280706	GQ267312	Crous et al. (2006)
Ca. queenslandica	CBS 112146T	CMW 30604 = CPC 3213	GQ280496	AF389835	GQ267415	FJ918521	GQ280618	GQ280740	FJ918543	Lombard et al. (2010c)
	CBS 112155	CMW 30603 = CPC 3210	GQ280497	AF389834	GQ267416	DQ190667	GQ280619	GQ280741	FJ918544
Ca. reteaudii	CBS 112143	CMW 16738 = CPC 3200	GQ280499	GQ240642	GQ267418	DQ190660	GQ280621	GQ280743	FJ918536	Crous (2002)
	CBS 112144T	CMW 30984 = CPC 3201	GQ280498	AF389833	GQ267417	DQ190661	GQ280620	GQ280742	FJ918537
Ca. rumohrae	CBS 109062	CMW 30989 = CPC 1603	GQ280501	AF232873	GQ267420	DQ190676	GQ280623	GQ280745	FJ918550	Crous (2002)
	CBS 111431T	CMW 23697 = CPC 1716	GQ280500	AF232871	GQ267419	DQ190675	GQ280622	GQ280744	FJ918549
Ca. scoparia	CMW 31000	CPC 1675 = UFV 117	GQ280435	FJ972426	GQ267367	FJ972476	GQ280557	GQ280679	FJ972525	Crous (2002)
	CMW 31001	UFV 126	GQ280436	FJ972427	GQ267368	GQ267246	GQ280558	GQ280680	GQ267246
Ca. spathiphylli	CBS 114540	CMW 16742	GQ280505	AF348214	GQ267424	AF348230	GQ280627	GQ280749	GQ267330	Crous (2002)
	CBS 116168	CMW 30997	GQ280506	FJ918512	GQ267425	FJ918530	GQ280628	GQ280750	FJ918561
Ca. spathulata	CBS 555.92	CMW 16744	GQ280508	GQ267215	GQ267427	GQ267261	GQ280630	GQ280752	GQ267331	Crous (2002)
	CBS 112689	CMW 16745	GQ280507	AF308463	GQ267426	FJ918524	GQ280629	GQ280751	FJ918554
Ca. sulawesiensis	CBS 125248	CMW 14857	GQ280516	GQ267223	GQ267435	GQ267272	GQ280638	GQ280760	GQ267343	This study
	CBS 125253	CMW 14879	GQ280513	GQ267220	GQ267432	GQ267269	GQ280635	GQ280757	GQ267340
	CBS 125277T	CMW 14878	GQ280515	GQ267222	GQ267434	GQ267271	GQ280637	GQ280759	GQ267342
	CMW 14883		GQ280514	GQ267221	GQ267433	GQ267270	GQ280636	GQ280758	GQ267341
Ca. sumatrensis	CBS 112829T	CMW 23698 = CPC4518	GQ280532	AY725649	AY725771	AY725696	GQ280654	GQ280776	AY725733	Crous et al. (2004b)
	CBS 112934	CMW 30987 = CPC 4516	GQ280533	AY725651	AY725773	AY725798	GQ280655	GQ280777	AY725735
Ca. terrae-reginae	CBS 112151T	CMW 30601 = CPC 3202	GQ280534	FJ918506	GQ267451	FJ918522	GQ280656	GQ280778	FJ918545	Lombard et al. (2010c)
	CBS 112634	CMW 30602 = CPC 4233	GQ280535	FJ918507	GQ267452	DQ190668	GQ280657	GQ280779	FJ918546
Ca. variabilis	CBS 112691	CMW 2914	GQ280541	GQ267240	GQ267458	GQ267264	GQ280663	GQ280785	GQ267335	Crous (2002)
	CBS 114677	CMW 3187	GQ280540	AF333424	GQ267457	GQ267263	GQ280662	GQ280764	GQ267334
Ca. zuluensis	CBS 125268	CMW 9188T	GQ280542	FJ972414	GQ267459	FJ972433	GQ280664	GQ280786	FJ972483	Lombard et al. (2010a)
	CBS 125272	CMW 9896	GQ280543	FJ972415	GQ267460	FJ972434	GQ280665	GQ280787	FJ972484

CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Pedro Crous working collection housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; ATCC: American Type Culture Collection, Virginia, U.S.A.; UFV: Univeridade Federal de Viçosa, Brazil. 2ACT = Actin, BT = β-tubulin, CAL = Calmodulin, HIS3 = Histone H3, ITS = Internal transcribed spacer regions 1 and 2 and the 5.8S gene of the ribosomal RNA, LSU = 28S large subunit RNA, TEF-1α = Translation elongation factor 1-alpha. 3References used for species descriptions. TEx-type cultures.

DNA extraction and amplification

Identification of unknown Calonectria isolates

Total genomic DNA was extracted from 7 d old Calonectria cultures using the methods presented in Lombard et al. (2008). Three loci were amplified and sequenced. These included a fragment of the BT gene region using primers T1 (O'Donnell & Cigelnik 1997) and CYLTUB1R (Crous ), a fragment of the HIS3 gene region using primers CYLH3F and CYLH3R (Crous ) and a fragment of the TEF-1α gene region using primers EF1-728F (Carbone & Kohn 1999) and EF2 (O'Donnell ).

Phylogenetic relationships amongst Calonectria spp.

Total genomic DNA was extracted as above. Seven loci were amplified including the ITS gene region using primers V9G (De Hoog & van den Ende 1998) and ITS4 (White ); the 28S large subunit RNA gene (LSU) using primers LR0R (Moncalvo ) and LR5 (Vilgalys & Hester 1990); and parts of the TEF-1α gene region; the BT gene region; the HIS3 gene region with the same primer sets mentioned previously, the actin (ACT) gene region using primers ACT-512F and ACT-783R (Carbone & Kohn 1999) and CAL gene region using primers CAL-228F and CAL-737R (Carbone & Kohn 1999).

The PCR reaction mixture used to amplify the different loci consisted of 2.5 units FastStart Taq polymerase (Roche Applied Science, USA), 1× PCR buffer, 1–1.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 μm of each primer and approximately 30 ng of fungal genomic DNA, made up to a total reaction volume of 25 μL with sterile deionised water. Amplified fragments were purified using High Pure PCR Product Purification Kit (Roche, U.S.A.).

DNA sequencing and analysis

Amplified fragments were sequenced in both directions using the same primer pairs used for amplification. For this purpose, the BigDye terminator sequencing kit v. 3.1 (Applied Biosystems, U.S.A.) and an ABI PRISM™ 3100 DNA sequencer (Applied Biosystems) were used. All PCRs and sequencing reactions were performed on an Eppendorf Mastercycler Personal PCR (Eppendorf AG, Germany) with cycling conditions as described in Crous et al. (2006) for all loci amplified.

In addition to the sequences generated in this study, Calonectria spp. sequences were obtained from GenBank. All sequences were assembled and aligned using Sequence Navigator v. 1.0.1 (Applied Biosystems) and MAFFT v. 5.11 (Katoh ), respectively. The aligned sequences were then manually corrected where necessary. Single nucleotide polymorphisms (SNP's) were determined for the aligned DNA sequences of each gene region using DnaSP v. 5.00.06 (Librado & Rozas 2009)

To determine whether the DNA sequence data sets were congruent, a partition homogeneity test (PHT; Farris ) of all possible combinations, with 1 000 replications on all informative characters was conducted in PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10; Swofford 2002). A 70 % reciprocal bootstrap method using Neighbour-Joining with Maximum Likelihood distance (Mason-Gamer & Kellogg 1996; Gueidan ) was also employed. Models of evolution were estimated in Modeltest v. 3.7 (Posada & Crandall 1998) using the Akaike Information Criterion (AIC) for each gene region. The bootstrap analyses were run in PAUP for 10 000 replicates. Resulting tree topologies were compared visually for conflict between the separate gene regions.

Maximum-parsimony genealogies, for single genes and the combined genes were estimated in PAUP, by heuristic searches based on 1 000 random addition sequences and tree bisection-reconnection, with the branch swapping option set on “best trees” only. All characters were weighted equally and alignment gaps were treated as missing data. Statistics calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistence index (RC). Bootstrap analysis (Hillis & Bull 1993) was based on 1 000 replications. All sequences for the isolates studied were analysed using the Basic Local Alignment Search Tool for Nucleotide sequences (BLASTN, Altschul ).

A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees for each gene region and combined sequence data subsets with Bayesian probabilities using MrBayes v. 3.1.1 (Ronquist & Huelsenbeck 2003). Models of nucleotide substitution for each gene were determined using MrModeltest (Nylander 2004) and included for each gene partition. Four MCMC chains were run simultaneously from random trees for one million generations, sampled every 100 generations and repeated twice. Both runs converged on the same likelihood score and tree topology for each gene. The first 1 000 trees were, therefore, discarded as the burn-in phase of each analysis and posterior probabilities were determined from the remaining trees.

Sexual compatibility

Based on the results of the DNA sequence analyses, single conidial isolates of Calonectria spp. of unknown identity were crossed with closely related species in all possible combinations. Where available, mating tester strains defined in previous studies were also used. Crosses were made as described in Schoch et al. (1999) on carnation leaf agar (CLA; Fisher , Crous ) and minimal salt agar (MSA; Guerber & Correll 2001, Halleen ) with sterile toothpicks placed on the surface of the agar (Lombard ). Controls consisted of isolates self-crossed, making it possible to distinguish between those having heterothallic or homothallic mating systems. Isolates CBS 125273–125276 from Indonesia were mated with Ca. macroconidialis (CBS 114880). Colombian isolates CBS 123698 and CMW 31210 and Indonesian isolates CBS 125258–125260 were crossed with Ca. brachiatica (CBS 123700 and CMW 25302) and Ca. brassicae (CBS 111478 and CBS 111869) in all possible combinations. Isolates CBS 125248, CBS 125253, CBS 125277 and CMW 14883 were crossed with Ca. cerciana (CBS 123693 and CBS 123695), Ca. brasiliensis (CBS 230.51 and CBS 114257) and mating tester strains of Ca. insularis (CBS 114558 and CBS 114559; Schoch ). Similarly, isolates CBS 125249–125252, CBS 125261 and CBS 125269 were crossed with mating tester strains of Ca. spathiphylli (CBS 114540 and CBS 116168; Crous 2002). Isolates CBS 125254–125257 were crossed with mating tester strains of Ca. scoparia (CMW 31000 and CMW 31001; Lombard ) and Ca. pauciramosa (CMW 5683 and CMW 30823; Schoch ). The plates were stacked in plastic containers and incubated at 22 °C for 6–8 wk. Crosses were regarded as successful when isolate combinations produced numerous perithecia extruding viable ascospores.

Taxonomy

For identification of Calonectria isolates based on morphology, single conidial cultures were prepared on MEA and synthetic nutrient-poor agar (SNA; Nirenburg 1981, Lombard et al. 2009, 2010a, c). Inoculated plates were incubated at room temperature and examined after 7 d. Gross morphological characteristics of the anamorph structures were determined by mounting fungal structures in lactic acid and 30 measurements at ×1 000 magnification were made for all taxonomically informative characters for each isolate. Teleomorph morphology was determined by mounting perithecia resulting from the sexual compatibility tests in Leica mountant (Setpoint Premier, Johannesburg, South Africa) and making sections using a Leica CM1100 cryostat (Setpoint Technologies) at -20 °C. The 10 μm sections were mounted in lactophenol or 3 % KOH. Gross morphological characteristics were determined in the same manner as for the anamorph states. The 95 % confidence levels were determined and extremes of conidial measurements are given in parentheses. For other structures, only extremes are presented in the descriptions. Optimal growth conditions for cultures were determined in the dark on MEA for each isolate, at temperatures ranging from 5–35 °C at 5 °C intervals with three replicate plates for each temperature tested. Two measurements of culture diameter perpendicular to each other were made daily for 7 d. Colony colours were determined after 7 d on MEA at 25 °C in the dark, using the colour charts of Rayner (1970). Descriptions, nomenclature and illustrations were deposited in MycoBank (Crous ).

RESULTS

Amplicons of approx. 500 bp were generated for the BT and TEF-1α gene regions and those for the HIS3 region were approx. 450 bp in length. Based on preliminary BT sequence comparisons and morphological characteristics, the sequence data sets for the unknown Calonectria spp. were divided into four separate data sets representing the Ca. colhounii, Ca. brassicae, Ca. scoparia and Ca. morganii complexes and other closely related species in each data set. These data sets were analysed separately with Ca. colombiensis (CBS 112221) and Ca. chinensis (CBS 112744) as outgroup taxa. For Bayesian analyses, a HKY+I+G model was selected for BT and TEF-1α, and GTR+I+G for HIS3 for all four data sets, which was incorporated in the analyses. The consensus trees obtained from the Bayesian analyses confirmed the tree topologies obtained with maximum-parsimony as well as bootstrap support. Therefore, only maximum-parsimony trees are presented with bootstrap values and posterior probabilities shown for well-supported branches.

The partition homogeneity tests for all possible combinations of the three gene regions used, consistently yielded a P-value of 0.001 for the four separate data sets. The 70 % reciprocal bootstrap trees showed no conflict in tree topologies for the three gene regions in each of the four separate data sets. Based on the tree topologies of the 70 % reciprocal bootstrap trees and a P-value of 0.001 in the PHT (Cunningham 1997, Dettman ) the DNA sequences for the three gene regions were combined for each of the four separate data sets.

The combined sequence data set representing the Ca. colhounii complex, with 10 taxa including outgroups, consisted of 1 497 characters, including gaps. Of these characters, 1 051 were constant, 133 were parsimony-uninformative and 313 characters were parsimony informative. Parsimony analysis of the aligned sequences yielded one most parsimonious tree (Fig. 1; TL = 649 steps; CI = 0.888; RI = 0.891; RC = 0.791). In the tree, isolates CBS 125273–125276, from Indonesia, grouped close to but separate from Ca. colhounii (CBS 293.79 and CBS 114704) with 100 % bootstrap support (BP) and a posterior probability (PP) of 0.97. The SNP analyses showed 16 unique alleles for the Indonesian isolates with one shared unique allele with Ca. madagascariensis (CBS 114571 and CBS 114572) and two shared alleles with Ca. macroconidialis (CBS 114880) for the three gene regions analysed (Table 2). These unique alleles, however, distinguish the Indonesian isolates from Ca. colhounii, Ca. macroconidialis and Ca. madagascariensis.

Fig. 1.

The most parsimonious trees obtained from a heuristic search with 1 000 random additions sequences of the combined BT, HIS3 and TEF-1α sequence alignments of the Ca. colhounii complex. Scale bar shows 10 changes and bootstrap support values (bold) from 1 000 replicates and Bayesian posterior probability values are indicated at the nodes. Red lines indicate bootstrap values of 100 and posterior probabilities of 1.00. Thickened lines indicate branches in the strict consensus tree and the consensus tree of the Bayesian analyses.

Table 2.

Single nucleotide polymorphisms comparisons between Ca. eucalypti and Ca. colhounii, compared to Ca. macroconidialis and Ca. madagascariensis.

The data set representing the Ca. brassicae complex consisted of 15 taxa including the outgroups, while the combined sequence alignment was made up of 1 509 characters, including gaps. These characters represented 1 092 constant, 127 parsimony-uninformative and 290 parsimony-informative characters. Parsimony analysis yielded one most parsimonious tree (Fig. 2; TL = 569 steps; CI = 0.931; RI = 0.918; RC = 0.855). In the tree, Colombian isolates CBS 123698 and CBS 125523 clustered close to Ca. brassicae (CBS 111869 and CBS 111478) and Ca. brachiatica (CBS 123700 and CMW 25302) but separately from both these species with high support (BP = 100 and PP = 1.00). Similarly, isolates CBS 125258–125260, from Indonesia, clustered together closely related to Ca. brassicae and Ca. brachiatica. These Indonesian isolates were also closely related to the Colombian isolates but grouped separately from them in a clade with high support (BP = 97 and PP = 1.00). The SNP analyses showed that isolates CBS 123698 and CBS 125523 have 18 unique alleles and isolates CBS 125258–125260 have four unique alleles distinguishing them from each other for the three gene regions analysed. These isolates also share 14 unique alleles, distinguishing them from Ca. brassicae and Ca. brachiatica (Table 3).

Fig. 2.

The most parsimonious trees obtained from a heuristic search with 1 000 random additions sequences of the combined BT, HIS3 and TEF-1α sequence alignments of the Ca. brassicae complex. Scale bar shows 10 changes and bootstrap support values (bold) from 1 000 replicates and Bayesian posterior probability values are indicated at the nodes. Red lines indicate bootstrap values of 100 and posterior probabilities of 1.00. Thickened lines indicate branches in the strict consensus tree and the consensus tree of the Bayesian analyses.

Table 3.

Single nucleotide polymorphisms from the sequence datasets for Ca. pini and Ca. orientalis compared to Ca. brachiatica and Ca. brassicae.

The third data set, represented by 16 ingroup taxa residing in the Ca. scoparia complex and closely related species, consisted of 1 530 characters including gaps for the three gene regions analysed. Of these characters, 1 114 were constant, 138 were parsimony-uninformative and 278 characters were parsimony informative. Parsimony analysis of the aligned sequences yielded two most parsimonious trees (TL = 551 steps; CI = 0.902; RI = 0.925; RC = 0.834), one of which is presented in Fig. 3. In the tree, isolates CBS 125254–125257 from Ecuador, clustered closely but separately from Ca. scoparia (CMW 31000 and CMW 31001) and other species in the Ca. pauciramosa complex with low support (BP = 63 and PP = 1.00). The Ecuadorian isolates also had three unique alleles separating them from Ca. scoparia and Ca. pauciramosa (CMW 5683 and CMW 30823) for the BT and TEF-1α regions, but there were no unique alleles for these isolates in the HIS3 region (Table 4).

Fig. 3.

One of two most parsimonious trees obtained from a heuristic search with 1 000 random additions sequences of the combined BT, HIS3 and TEF-1α sequence alignments of the Ca. scoparia complex. Scale bar shows 10 changes and bootstrap support values (bold) from 1 000 replicates and Bayesian posterior probability values are indicated at the nodes. Red lines indicate bootstrap values of 100 and posterior probabilities of 1.00. Thickened lines indicate branches in the strict consensus tree and the consensus tree of the Bayesian analyses.

Table 4.

Single nucleotide polymorphisms comparisons between Ca. scoparia and Ca. pseudoscoparia, compared to Ca. pauciramosa.

The aligned sequence data set for the Ca. morganii complex included 25 ingroup taxa consisting of 1 535 characters. Of these characters, 975 were constant, 211 were parsimony-uninformative and 349 characters were parsimony-informative. Parsimony analysis of the aligned sequences yielded three most parsimonious trees (TL = 977 steps; CI = 0.784; RI = 0.825; RC = 0.647), one of which is presented in Fig. 4. In the tree, isolates CBS 125249–125252, CBS 125261 and CBS 125269 from Ecuador clustered in a clade (BP = 99 and PP = 1.00) with Ca. spathiphylli (CBS 114540 and CBS 116168) and Ca. pseudospathiphylli (CBS 109165), whereas isolates CBS 125248, CBS 125253, CBS 125277 and CMW 14883 from Indonesia clustered close to Ca. brasiliensis (CBS 230.51 and CBS 114257) but with low support (BP = 52; PP = 0.90) in a separate, well-supported clade (BP = 100; PP = 1.00). Isolates CBS 125249, CBS 125250 and CBS 125261 clustered together in a well-supported clade (BP = 93; PP = 1.00) separate from CBS 125251, CBS 125252 and CBS 125269, that also clustered together in a well-supported clade (BP = 81; PP = 1.00). Both clades were separate from Ca. spathiphylli and Ca. pseudospathiphylli but closely related to these species. The SNP analyses showed that isolates CBS 125249, CBS 125250 and CBS 125261 shared four unique alleles and CBS 125251, CBS 125252 and CBS 125269 shared seven unique alleles for the three gene regions. These isolates also shared an additional 33 alleles, distinguishing them from Ca. spathiphylli (Table 5). Isolates CBS 125248, CBS 125253, CBS 125277 and CMW 14883 shared eight unique alleles, distinguishing them from Ca. brasiliensis (CBS 230.51 and CBS 114257), Ca. cerciana (CBS 123693 and CBS 123695) and Ca. insularis (CBS 114558 and CBS 114559) (Table 6).

Fig. 4.

One of three most parsimonious trees obtained from a heuristic search with 1 000 random additions sequences of the combined BT, HIS3 and TEF-1α sequence alignments of the Ca. morganii complex. Scale bar shows 10 changes and bootstrap support values (bold) from 1 000 replicates and Bayesian posterior probability values are indicated at the nodes. Red lines indicate bootstrap values of 100 and posterior probabilities of 1.00. Thickened lines indicate branches in the strict consensus tree and the consensus tree of the Bayesian analyses.

Table 5.

Single nucleotide polymorphisms from the sequence datasets for Ca. densa and Ca. humicola compared to Ca. spathiphylli.

Table 6.

Single nucleotide polymorphisms comparisons between Ca. brasiliensis, Ca. insularis and Ca. sulawesiensis compared to Ca. cerciana.

Approximately 250 bases were determined for ACT, 450 bases for HIS3, 500 for BT, CAL and TEF-1α, 700 for ITS and 880 for LSU. The adjusted sequence alignments for each gene region consisted of 122 ingroup taxa with Cylindrocladiella lageniformis (CBS 112898) and C. peruviana (CPC 5614) as outgroup taxa for each gene region. For Bayesian analyses, a K80+G model was selected for ACT, HKY+I+G for BT, CAL and TEF-1α, GTR+I+G for HIS3 and LSU, and SYM+I+G for ITS and incorporated in the analyses. The consensus trees obtained from the Bayesian analyses confirmed the tree topologies obtained with maximum-parsimony as well as bootstrap support.

Individual analyses of the gene regions showed similar tree topologies for the protein coding regions (ACT, BT, CAL, HIS3 and TEF-1α) with well-supported clades for Calonectria spp. with similar morphological characteristics. In contrast, the non-coding gene regions (ITS and LSU) provided little or no support for the clades that emerged from the protein coding regions, with several Calonectria spp. clustering together with no significant similarities. The trees for the ITS and LSU regions showed a single monophyletic clade for all Calonectria spp. and did not reveal the two clades observed for the coding gene regions. The phylogeny constructed based on CAL sequences showed the best resolution of the species and it had the highest support for the individual clades, followed by TEF-1α gene region. Statistical data for the individual trees (not shown) are presented in Table 7.

Table 7.

Statistical information on the sequence dataset and maximum parsimony trees for each locus.

	Actin	β-tubulin	Calmodulin	Histone H3	ITS	LSU	TEF-1α
Aligned characters	290	532	531	499	706	887	596
Variable characters	15	42	39	62	32	10	57
Informative characters	151	268	323	223	112	37	337
Most parsimonious trees	2622	91	1000	372	1000	100	9970
Tree length	573	1454	1282	1843	296	91	1641
CI	0.490	0.431	0.467	0.352	0.618	0.538	0.477
RI	0.867	0.840	0.849	0.793	0.882	0.913	0.871
RC	0.425	0.569	0.397	0.648	0.545	0.492	0.416

The partition homogeneity tests for all possible combinations of the seven gene regions used, consistently yielded a P-value of 0.001. The 70 % reciprocal bootstrap trees showed no conflict in tree topologies for the five coding gene regions (ACT, BT, CAL, HIS3 and TEF-1α), however conflicts were observed between the non-coding gene regions (ITS and LSU) and the coding gene regions. Based on the tree topologies and a P-value of 0.001 (Cunningham 1997, Dettman ) the sequence data for coding gene regions were combined. The data for the ITS and LSU datasets were treated separately, but these are not presented because they add little taxonomic value. However, all ITS and LSU sequences generated in this study have been deposited in GenBank and TreeBase (Table 1).

The combined sequence alignment of the five coding gene regions consisted of 2 472 characters, including gaps. Of these characters, 925 were constant, 267 were parsimony-uninformative and 1 280 characters were parsimony-informative. Parsimony analysis of the aligned sequences yielded 24 most parsimonious trees (TL = 7319 steps; CI = 0.397; RI = 0.820; RC = 0.326), one of which is presented in Fig. 5. The tree topology obtained with the combined sequence dataset was similar to that obtained for the individual gene regions analysed and therefore the only tree presented is that of the combined dataset.

Fig. 5.

One of 24 most parsimonious trees obtained from a heuristic search with 1 000 random additions sequences of the combined actin, β-tubulin, calmodulin, histone H3 and translation elongation factor 1-alpha sequence alignments of the Calonectria. Scale bar shows 10 changes and bootstrap support values (bold) from 1 000 replicates and Bayesian posterior probability values are indicated at the nodes. Thickened lines indicate branches in the strict consensus tree and the consensus tree of the Bayesian analyses. Red lines indicate bootstrap values of 100 and posterior probabilities of 1.00. The tree was rooted to Cylindrocladiella lageniformis (CBS 112898) and C. peruviana (CPC 5614). Phylogenetic groups are indicated on the right.

In the tree (Fig. 5), the Calonectria spp. were found to clearly reside in two main clades which was consistent for the analyses for these gene regions separately. One of these clades (BP = 82, PP = 0.62) which we refer to as representing the Prolate Group, includes Calonectria spp. with clavate to pyriform to ellipsoidal vesicles. This clade (Fig. 5) is made up of two sub-clades, one (BP = 81, PP = 1.00) of which includes 10 minor clades representing Calonectria spp. that have vesicles and conidia that have similar morphology. The second sub-clade (BP = 99, PP = 1.00) representing the Prolate Group includes taxa represented by single isolates and for which there were no obvious unifying morphological characters.

The second main clade (BP = 65, PP = 0.64) which is referred to as the Sphaero-Naviculate Group of species included Calonectria spp. characterised by sphaeropedunculate and naviculate vesicles and these were also seen in the analyses based on the individual gene regions. This clade is further sub-divided into two clades. The first of these sub-clades (BP = 65, PP = 1.00) includes Calonectria spp. characterised by sphaeropedunculate vesicles. The second sub-clade (BP = 93, PP = 0.86) accommodates Calonectria spp. with naviculate vesicles.

The only isolates in the mating tests that yielded perithecia were CBS 125273–125276 (Fig. 6). These isolates all produced perithecia containing viable ascospores within 6 wk when mated with themselves, indicating that they are self-fertile (homothallic). All other control inoculations with the selected isolates failed to yield perithecia, indicating that they were either self-sterile (heterothallic) and non-compatible, or that they had lost the ability to undergo sexual recombination.

Fig 6.

Results of sexual compatibility tests. Successful matings are indicated by (+) and unsuccessful matings is indicated with (-). Blue highlighted blocks indicate homothallic matings. Yellow blocks highlight unsuccessful self-self matings. Purple blocks indicate mating tester strain matings. A. Matings between isolates of Ca. macroconidialis and Ca. eucalypti. B. Matings between isolates of Ca. brachiatica, Ca. brassicae, Ca. pini and Ca. orientalis. C. Matings between isolates of Ca. brasiliensis, Ca. cerciana, Ca. insularis and Ca. sulawesiensis. D. Matings between isolates of Ca. densa, Ca. humicola and Ca. spathiphylli. E. Matings between isolates of Ca. pauciramosa, Ca. pseudoscoparia and Ca. scoparia.

Based on morphological observations, phylogenetic inference and mating, numerous isolates of Calonectria spp. included in this study represent undescribed species. Species of Cylindrocladium (1892) represent anamorph states of Calonectria (1867) (Rossman ). In an attempt to move to a single nomenclature for pleomorphic fungi, the teleomorph name takes precedence over the anamorph name when both types belong to the same holomorph. The species below are described as new species in Calonectria, which represents the older generic name for these holomorphs and follows Lombard et al. (2009, 2010a, c). All Cylindrocladium species without a Calonectria state, are also transferred to Calonectria.

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515529, Fig. 7.

Fig. 7.

Calonectria densa. A–D. Macroconidiophores. E–I. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. J–L. Ovoid to ellipsoid vesicles. M–N. One-septate macroconidia. Scale bars = 10 μm.

Etymology: Name refers to the fact that lateral stipe extensions are readily formed in this species, giving it a bushy appearance.

Teleomorpha ignota. Anamorpha Cy. spathiphylli similis sed extensiones laterales stiparum facit, macroconidiis cylindricis utrinque rotundatis rectis (47–)50–58(–62) × 5–6 μm mediocriter 54 × 6 μm, semel septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis differ.

Teleomorph unknown. Conidiophores with a stipe bearing penicillate suites of fertile branches, stipe extensions, and terminal vesicles; stipe septate, hyaline, smooth, 54–90 × 6–10 μm; stipe extensions septate, straight to flexuous, 149–192 μm long, 5–6 μm wide at the apical septum, terminating in ovoid to ellipsoid to sphaeropedunculate vesicles, 10–12 μm diam; lateral stipe extensions (90° to the axis) also present. Conidiogenous apparatus 49–78 μm long, and 63–123 μm wide; primary branches aseptate, 20–29 × 5–6 μm; secondary branches aseptate, 16–20 × 4–6 μm; tertiary and additional branches (–4) aseptate, 9–16 × 3–5 μm, each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, 11–16 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (47–)50–58(–62) × (5–)6 μm (av. = 54 × 6 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not seen.

Specimens examined: Ecuador, Pichincha Province, Las Golondrinas, from soil, Dec. 2004, M.J. Wingfield, Herb. PREM 60302, holotype of Ca. densa, culture ex-type CMW 31182 = CBS 125261; Pichincha Province, Las Golondrinas, from soil, Dec. 2004, M.J. Wingfield, cultures CMW 31184 = CBS 125249; Las Golondrinas, from soil, Dec. 2004, M.J. Wingfield, culture CMW 31185 = CBS 125250.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 15–35 °C) on MEA, reverse umber to verona-brown after 7 d; moderate white aerial mycelium with moderate sporulation; chlamydospores extensive throughout the medium forming microsclerotia.

Substrate: Soil.

Distribution: Ecuador.

Notes: Morphologically, Ca. densa is very similar to Ca. spathiphylli and Ca. pseudospathiphylli. However, macroconidia of Ca. densa (av. 54 × 6 μm) are smaller than those of Ca. spathiphylli (av. 70 × 6 μm), but slightly larger and broader than those of Ca. pseudospathiphylli (av. 52 × 4 μm). Calonectria densa also readily forms lateral stipe extensions, not reported for the other two species.

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515530, Fig. 8.

Fig. 8.

Calonectria eucalypti. A. Perithecium. B. Section through ostiolar region of a perithecium. C. A vertical section through a perithecium, showing wall layers. D. Ascus. E–G. Ascospores. H–L. Macroconidiophores. M–P. Conidiogenous apparatus with conidiophore branches and doliiform to reniform or allantoid phialides. Q–U. Clavate to broadly clavate vesicles. V–W. Three-septate macroconidia. Scale bars: A = 90 μm, H–I = 70 μm, Other bars = 10 μm.

Etymology: Name refers to Eucalyptus from which the fungus was isolated.

Teleomorpha Ca. colhounii similis sed ascocarpo flavo vel aurantiaco differt. Anamorpha Cy. colhounii similis sed macroconidiis cylindricis utrinque rotundatis rectis (66–)69–75(–80) × 5–6 μm mediocriter 72 × 6 μm, ter septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis, differt.

Perithecia solitary or in groups, yellow to orange, becoming brown with age; in section apex and body yellow to orange, base red-brown, sub-globose to ovoid, 325–510 μm high, 285–360 μm diam, body turning dark red, and base dark red-brown (KOH+). Perithecial walls rough consisting of 2 thick-walled layers: outside layer of textura globulosa, 45–90 μm wide; becoming more compressed towards inner layer of textura angularis, 12–18 μm wide; becoming thin-walled and hyaline towards the centre, outer cells 24–50 × 10–40 μm; inner cells 6–19 × 3–6 μm: perithecial base up to 125 μm wide; consisting of dark red, angular cells; merging with an erumpent stroma, cells of the outer wall layer continuing into the pseudoparenchymatous cells of the erumpent stroma. Asci 4-spored, clavate, 92–188 × 10–27 μm, tapering to a long thin stalk. Ascospores aggregated in the upper third of the ascus, hyaline, guttulate, fusoid with rounded ends, straight to slightly curved, (1–)3-septate, not or slightly constricted at the septum, (25–)30–36(–56) × (3–)5–6(–8) μm (av. = 33 × 6 μm). Cultures were homothallic. Conidiophores with a stipe bearing a suit of penicillate, fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, 45–91 × 7–10 μm; stipe extensions septate, straight to flexuous, 110–235 μm long, 5–6 μm wide at the apical septum, terminating in broadly clavate vesicles, 4–6 μm diam. Conidiogenous apparatus 52–82 μm long, and 40–95 μm wide; primary branches aseptate or 1-septate, 21–29 × 5–6 μm; secondary branches aseptate, 14–21 × 3–5 μm; tertiary branches and additional branches (–5), aseptate, 11–16 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 10–14 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (66–)69–75(–80) × (5–)6 μm (av. = 72 × 6 μm), 3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Megaconidia and microconidia not seen.

Specimens examined: Indonesia, Sumatra Utara, Aek Nauli, on leaf of Eucalyptus grandis, May 2005, M.J. Wingfield, Herb. PREM 60298, holotype of Ca. eucalypti, culture ex-type CMW 18444 = CBS 125275; Aek Nauli, on leaf of Eucalyptus grandis, May 2005, M.J. Wingfield, PREM 60299, culture CMW 14890 = CBS 125273; Aek Nauli, on leaf of Eucalyptus grandis, May 2005, M.J. Wingfield, culture CMW 18443 = CBS 125274, Aek Nauli, on leaf of Eucalyptus grandis, May 2005, M.J. Wingfield, culture CMW 18445 = CBS 125276.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 15–30 °C) on MEA, reverse colour tawny-brown after 7 d; abundant white aerial mycelium and sporulation; chlamydospores abundant throughout the medium, forming microsclerotia.

Substrate: Eucalyptus grandis.

Distribution: Indonesia.

Notes: The perithecia of Ca. eucalypti can be distinguished from Ca. colhounii and Ca. macroconidialis based on their yellow to orange colour in KOH. Macroconidia of Ca. eucalypti (av. 72 × 6 μm) are also larger than those of Ca. colhounii (av. 55 × 6 μm) and Ca. madagascariensis (av. 55 × 4.5 μm), but smaller than those of Ca. macroconidialis (av. 90 × 6.5 μm). Mating tests (Fig. 6) also showed that Ca. eucalypti is homothallic, a characteristic shared by Ca. colhounii and Ca. madagascariensis but not with Ca. macroconidialis, which is heterothallic (Crous 2002).

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515531, Fig. 9.

Fig. 9.

(p. 50) Calonectria humicola. A–F. Macroconidiophores. G–I. Conidiogenous apparatus with conidiophore branches and somewhat elongated, doliiform to reniform phialides. J–N. Globose to ovoid to sphaeropedunculate vesicles. O–P. One-septate macroconidia. Scale bars = 10 μm.

Etymology: Name refers to the fact that this fungus was isolated from soil.

Teleomorpha ignota. Anamorpha Cy. spathiphylli similis sed macroconidiis cylindricis utrinque rotundatis rectis (45–)48–54(–56) × 4–5 μm mediocriter 51 × 5 μm, semel septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis differt.

Teleomorph unknown. Conidiophores with a stipe bearing penicillate suites of fertile branches, stipe extensions, and terminal vesicles; stipe septate, hyaline, smooth, 44–90 × 6–8 μm; stipe extensions septate, straight to flexuous, 126–157 μm long, 4–5 μm wide at the apical septum, terminating in globose to ovoid to sphaeropedunculate vesicles, 10–12 μm diam. Conidiogenous apparatus 43–71 μm long, and 42–49 μm wide; primary branches aseptate, 20–29 × 4–6 μm; secondary branches aseptate, 12–19 × 3–5 μm; tertiary branches aseptate, 9–16 × 3–5 μm, each terminal branch producing 2–4 phialides; phialides elongated doliiform to reniform, hyaline, aseptate, 10–15 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (45–)48–54(–56) × (4–)5 μm (av. = 51 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not seen.

Specimens examined: Ecuador, Pichincha Province, Las Golondrinas, from soil, Dec. 2004, M.J. Wingfield, Herb. PREM 60369 holotype of Ca. humicola, culture ex-type CMW 31183 = CBS 125251; Las Golondrinas, from soil, Jan. 2006, L. Lombard, culture CMW 31186 = CBS 125252; Las Golondrinas, from soil, Jan. 2006, L. Lombard, (Herb. PREM 60368) culture CMW 31187 = CBS 125269.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 15–35 °C) on MEA, reverse umber to verona-brown after 7 d; moderate white aerial mycelium with moderate sporulation; chlamydospores extensive throughout the medium, forming microsclerotia.

Substrate: Soil.

Distribution: Ecuador.

Notes: Calonectria humicola is morphologically very similar to Ca. densa, Ca. pseudospathiphylli and Ca. spathiphylli. However, no lateral stipe extensions occur in this species, whereas these are common in Ca. densa. Macroconidia of Ca. humicola (av. 51 × 5 μm) are slightly smaller than those of Ca. densa (av. 54 × 6 μm) and Ca. spathiphylli (av. 70 × 6 μm), but slightly broader than those of Ca. pseudospathiphylli (av. 52 × 4 μm).

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515532, Fig. 10.

Fig. 10.

Calonectria orientalis. A–C. Macroconidiophores. D–K. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. L–Q. Clavate vesicles. R–S. One-septate macroconidia. Scale bars = 10 μm.

Etymology: Name refers to the East Asian region, where the fungus was isolated.

Teleomorpha ignota. Anamorpha Ca. brachiaticae similis sed ramis conidiophorae tres vel minus sine extensionibus lateralibus stipae, macroconidiis cylindricis utrinque rotundatis rectis (43–)46–50(–53) × 4–5 μm mediocriter 48 × 4 μm, semel septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis differt.

Teleomorph unknown. Conidiophores with a stipe bearing penicillate suites of fertile branches, stipe extensions, and terminal vesicles; stipe septate, hyaline, smooth, 60–169 × 6–12 μm; stipe extensions septate, straight to flexuous, 90–218 μm long, 5–10 μm wide at the apical septum, terminating in clavate to broadly clavate vesicles, 5–10 μm diam. Conidiogenous apparatus 54–174 μm long, and 67–92 μm wide; primary branches aseptate, 19–30 × 4–7 μm; secondary branches aseptate, 16–29 × 4–6 μm; tertiary and additional branches (–5) aseptate, 10–20 × 5–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 10–19 × 2–5 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (43–)46–50(–53) × 4(–5) μm (av. = 48 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not seen.

Specimens examined: Indonesia, Langam, from soil, June 2005, M.J. Wingfield, Herb. PREM 60303, holotype of Ca. orientalis, culture ex-type CMW 20291 = CBS 125260; Teso East, from soil, June 2005, M.J. Wingfield culture CMW 20273 = CBS 125259; Teso East, from soil, June 2005, M.J. Wingfield, culture CMW 20272 = CBS 125258.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 15–35 °C) on MEA, reverse sepia-brown after 7 d; abundant white aerial mycelium with moderate to extensive sporulation; chlamydospores extensive throughout the medium, forming microsclerotia.

Substrate: Soil.

Distribution: Indonesia.

Notes: Calonectria orientalis is closely related to Calonectria spp. in the Ca. brassicae complex, based on phylogenetic inference and SNP analyses. Morphological comparisons showed that the macroconidia of Ca. orientalis (av. 48 × 4 μm) are shorter than those of Ca. brassicae (av. 53 × 4.5 μm), Ca. clavata (av. 65 × 5 μm) and Ca. gracilis (av. 56 × 4.5 μm) but larger than those of Ca. brachiatica (av. 44 × 5 μm) and Ca. gracilipes (av. 45 × 4.5 μm). As with Ca. pini, perithecia could not be induced when this species was mated with Ca. brachiatica and Ca. brassicae, highlighting the rarity of teleomorph structures for this group of fungi.

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515533, Fig. 11.

Fig. 11.

Calonectria pini. A–E. Macroconidiophores. F–M. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. N–R. Clavate vesicles. S–T. One-septate macroconidia. Scale bars = 10 μm.

Etymology: Name refers to Pinus, the host from which the fungus was isolated.

Teleomorpha ignota. Anamorpha Ca. brachiaticae similis sed ramis conidiophorae tres vel minus sine extensionibus lateralibus stipae, macroconidiis cylindricis utrinque rotundatis rectis (37–)40–48(–50) × 4–6 μm mediocriter 44 × 5 μm, semel septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis differ.

Teleomorph unknown. Conidiophores with a stipe bearing penicillate suites of fertile branches, stipe extensions, and terminal vesicles; stipe septate, hyaline, smooth, 40–99 × 6–7 μm; stipe extensions septate, straight to flexuous, 121–266 μm long, 5–7 μm wide at the apical septum, terminating in clavate vesicles, 4–6 μm diam. Conidiogenous apparatus 49–81 μm long, and 35–84 μm wide; primary branches aseptate, 20–30 × 4–6 μm; secondary branches aseptate, 13–22 × 3–5 μm; tertiary branches aseptate, 11–15 × 3–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 10–15 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (37–)40–48(–50) × 4–6 μm (av. = 44 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not seen.

Specimens examined: Colombia, Valle del Cauca, Buga, from Pinus patula, Sept. 2007, C.A. Rodas, Herb. PREM 60304, holotype of Ca. pini, culture ex-type CMW 31209 = CBS 123698; Buga, from Pinus patula, Sept. 2007, C.A. Rodas; Buga, from Pinus patula, Sept. 2007, C.A. Rodas, culture CMW 31210 = CBS 125523.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 15–30 °C) on MEA, reverse amber to sepia-brown after 7 d; abundant white aerial mycelium with moderate to extensive sporulation; chlamydospores extensive throughout the medium forming microsclerotia.

Substrate: Pinus patula.

Distribution: Colombia.

Notes: Calonectria pini is very similar to Ca. brachiatica, but can be distinguished morphologically by the fact that it has three or fewer conidiophore branches and no lateral stipe extensions (Lombard ). Macroconidia of Ca. pini (av. 44 × 5 μm) are shorter than those of Ca. brassicae (av. 53 × 4.5 μm), Ca. gracilis (56 × 4.5 μm) and Ca. orientalis (av. 48 × 4 μm). This species also has fewer conidiophore branches than those mentioned above. Calonectria pini failed to produce perithecia when crossed with Ca. brachiatica and Ca. brassicae. This supports the findings of Crous et al. (2004b) and Lombard et al. (2009), that teleomorph structures are rarely observed in members of the Ca. brassicae complex.

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515534, Fig. 12.

Fig. 12.

Calonectria pseudoscoparia. A–E. Macroconidiophores. F–L. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. M–R. Obpyriform to ellipsoidal vesicles. S–T. One-septate macroconidia. Scale bars = 10 μm.

Etymology: Name reflects the fact that the species resembles the anamorph state of Ca. scoparia.

Teleomorpha ignota. Anamorpha Ca. scopario similis sed phialidibus elongato-doliiformibus vel reniformibus hyalinis non septatis 7–11 × 2–4 μm apice minute periclinale incrassatis colliculo inconspicuo, macroconidiis cylindricis utrinque rotundatis rectis (41–)45–51(–52) × 3–5 μm mediocriter 48 × 4 μm, semel septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis differ.

Teleomorph unknown. Conidiophores with a stipe bearing penicillate suites of fertile branches, stipe extensions, and terminal vesicles; stipe septate, hyaline, smooth, 56–107 × 6–10 μm; stipe extensions septate, straight to flexuous, 124–201 μm long, 4–6 μm wide at the apical septum, terminating in obpyriform to ellipsoidal vesicles, 6–10 μm diam. Conidiogenous apparatus 34–87 μm long, and 52–74 μm wide; primary branches aseptate, 26–38 × 4–7 μm; secondary branches aseptate, 17–28 × 4–6 μm; tertiary branches and additional branches (–4) aseptate, 14–19 × 3–4 μm, each terminal branch producing 2–6 phialides; phialides elongate-doliiform to reniform, hyaline, aseptate, 7–11 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (41–)45–51(–52) × 3–5 μm (av. = 48 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not seen.

Specimens examined: Ecuador, Pichincha Province, Las Golondrinas, Buenos Aires Nursery, from Eucalyptus grandis cutting, Dec. 2004, M.J. Wingfield, Herb. PREM 60305, holotype of Ca. pseudoscoparia, culture ex-type CMW 15218 = CBS 125257; Buenos Aires Nursery, from Eucalyptus grandis cutting, Dec. 2004, M.J. Wingfield, Herb. PREM 60306, cultures from different cuttings, CMW 15214 = CBS 125254, CMW 15215 = CBS 125255, CMW 15216 = CBS 125256.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 10–30 °C) on MEA, reverse amber to sepia-brown after 7 d; colony margins irregular with sparse to moderate white aerial mycelium with moderate sporulation; chlamydospores extensive throughout the medium forming microsclerotia.

Substrate: Eucalyptus grandis.

Distribution: Ecuador.

Notes: Calonectria pseudoscoparia (conidia av. 48 × 4 μm) can be distinguished from Ca. scoparia (conidia av. 60 × 4.5 μm) based on smaller macroconidia and the fact that it has elongated-doliiform to reniform phialides unlike those of Ca. pauciramosa and Ca. scoparia. Mating tests between this fungus and Ca. scoparia and Ca. pauciramosa failed to produce perithecia. Control crosses with both Ca. pauciramosa (CMW 5683 and CMW 30823) and Ca. scoparia tester isolates (CMW 31000 and CMW 31001) produced perithecia with viable ascospores showing that culture conditions were appropriate for mating.

L. Lombard, M.J. Wingf. & Crous, sp. nov. MycoBank MB515535, Fig. 13.

Fig. 13.

Calonectria sulawesiensis. A–E. Macroconidiophores. F–K. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. L–Q. Clavate to ellipsoidal vesicles. R–S. One-septate macroconidia. Scale bars = 10 μm.

Etymology: Name refers to the Indonesian island of Sulawesi, where the fungus was collected.

Teleomorpha ignota. Anamorpha Ca. morganii similis sed vesiculo terminali late clavato vel ellipsoideo 5–7 μm diametro, macroconidiis cylindricis utrinque rotundatis rectis (41–)45–51(–54) × (3–)4–6 μm mediocriter 48 × 4 μm, semel septatis, sine cicatrice abscissionis manifesta, cum muco hyalino in fasciculis parallelis cylindricis differt.

Teleomorph unknown. Conidiophores with a stipe bearing penicillate suites of fertile branches, stipe extensions, and terminal vesicles; stipe septate, hyaline, smooth, 37–139 × 5–11 μm; stipe extensions septate, straight to flexuous, 113–262 μm long, 5–7 μm wide at the apical septum, terminating in broadly clavate to ellipsoidal vesicles, 5–7 μm diam. Conidiogenous apparatus 41–79 μm long, and 43–81 μm wide; primary branches aseptate, 17–41 × 3–6 μm; secondary branches aseptate, 10–27 × 3–6 μm; tertiary branches and additional branches (–5), aseptate, 9–15 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 9–15 × 2–5 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (41–)45–51(–54) × (3–)4(–6) μm (av. = 48 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not seen.

Specimens examined: Indonesia, Sulawesi, from leaf of Eucalyptus sp., July 2003, M.J. Wingfield, Herb. PREM 60300, holotype of Ca. sulawesiensis, culture ex-type CMW 14878 = CBS 125277; Sulawesi, from leaf of Eucalyptus sp., July 2003, M.J. Wingfield, PREM 60301 culture CMW 14883; from different leaves, culture CMW 14859 = CBS 125248, CMW 14879 = CBS 125253.

Culture characteristics: Colonies fast growing with optimal growth temperature at 25 °C (growth at 15–30 °C) on MEA, reverse amber to sepia-brown after 7 d; abundant white aerial mycelium with moderate to extensive sporulation; chlamydospores extensive throughout the medium, forming microsclerotia.

Substrate: Eucalyptus sp.

Distribution: Indonesia.

Notes: There are a few morphological differences distinguishing Ca. sulawesiensis from other species in the Ca. morganii complex. Macroconidia of Ca. sulawesiensis (av. 48 × 4 μm) are slightly larger than those of Ca. brasiliensis (av. 30 × 4 μm), Ca. cerciana (av. 44 × 5 μm), Ca. insularis (av. 45 × 4 μm) and Ca. morganii (av. 45 × 4 μm), but smaller than those of Ca. hawksworthii (av. 56 × 4 μm), Ca. leucothoës (av. 73 × 5 μm) and Ca. variabilis (av. 73 × 5 μm). Mating tests where Ca. sulawesiensis was crossed with Ca. brasiliensis, Ca. cerciana and Ca. insularis failed to produce perithecia, or produced perithecia without viable ascospores.

(Crous & El-Gholl) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515536. Basionym: Cylindrocladium angustatum Crous & El-Gholl, Mycoscience 41: 522. 2000.

(Crous & K.D. Hyde) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515537. Basionym: Cylindrocladium australiense Crous & K.D. Hyde, Stud. Mycol. 55: 221. 2006.

(J.C. Kang, Crous & C.L. Schoch) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515538. Basionym: Cylindrocladium canadense J.C. Kang, Crous & C.L. Schoch, Syst. Appl. Microbiol. 24: 210. 2001.

(Crous) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515539. Basionym: Cylindrocladium chinense Crous, Stud. Mycol. 50: 420. 2004.

(H.S. Fawc. & Klotz) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515540. Basionym: Candelospora citri H.S. Fawc. & Klotz, Mycologia 29: 213. 1937.

≡ Cylindrocladium citri (H.S. Fawc. & Klotz) Boedijn & Reitsma, Reinwardtia 1: 57. 1950.

(Boedijn & Reitsma) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515541. Basionym: Cylindrocladium curvatum Boedijn & Reitsma, Reinwardtia 1: 54. 1950.

(Crous & D. Victor) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515542. Basionym: Cylindrocladium curvisporum Crous & D. Victor, Syst. Appl. Microbiol. 20: 283. 1997.

(Crous & M.J. Wingf.) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515543. Basionym: Cylindrocladium ecuadoriae Crous & M.J. Wingf., Stud. Mycol. 55: 222. 2006.

(Leahy, T.S. Schub. & El-Gholl) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515544. Basionym: Cylindrocladium gordoniae Leahy, T.S. Schub. & El-Gholl, Mycotaxon 76: 80. 2000.

(Peerally) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515545. Basionym: Cylindrocladium hawksworthii Peerally, Mycotaxon 40: 375. 1991.

(Linder & Whetzel) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515546. Basionym: Cercosporella hurae Linder & Whetzel, Mycologia 29: 656. 1937.

≡ Cylindrocladiopsis hurae (Linder & Whetzel) U. Braun, Mycotaxon 51: 40. 1994.

≡ Cylindrocladium hurae (Linder & Whetzel) Crous, In: Taxonomy and pathology of Cylindrocladium (Calonectria) and allied genera: 185. 2002.

= Cylindrocladium heptaseptatum Sober, Alfieri & Knauss, Phytopathology 65: 333. 1975.

= Cylindrocladiopsis lagerstroemiae J.M. Yen, Mycotaxon 8: 236. 1979.

(Crous) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515547. Basionym: Cylindrocladium indonesiae Crous, Stud. Mycol. 50: 424. 2004.

(El-Gholl, Leahy & T.S. Schub.) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515548. Basionym: Cylindrocladium leucothoës El-Gholl, Leahy & T.S. Schub., Canad. J. Bot. 67: 2530. 1989.

= Cylindrocladium perseae T.S. Schub., Leahy & El-Gholl, Mycotaxon 73: 474. 1999.

(Crous) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515549. Basionym: Cylindrocladium malesianum Crous, Stud. Mycol. 50: 425. 2004.

(Crous, Simoneau & Risède) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515550. Basionym: Cylindrocladium multiphialidicum Crous, Simoneau & Risède, Stud. Mycol. 50: 425. 2004.

(J.C. Kang, Crous & C.L. Schoch) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515551. Basionym: Cylindrocladium pacificum J.C. Kang, Crous & C.L. Schoch, Syst. Appl. Microbiol. 24: 213. 2001.

(Tubaki) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515552. Basionym: Candelospora penicilloides Tubaki, Nogaoa 2: 58. 1952.

≡ Cylindrocladium penicilloides (Tubaki) Tubaki, J. Hattori Bot. Lab. 20: 154. 1958.

(Crous, J.Z. Groenew. & C.F. Hill) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515554. Basionym: Cylindrocladium pseudonaviculatum Crous, J.Z. Groenew. & C.F. Hill, Sydowia 54: 26. 2002.

= Cylindrocladium buxicola Henricot, Mycologia 94: 993. 2002.

(Crous) L. Lombard, M.J. Wingf. & Crous, comb. nov. MycoBank MB515555. Basionym: Cylindrocladium sumatrense Crous, Stud. Mycol. 50: 426. 2004.

DISCUSSION

In this study, a collection of isolates of unknown identity were shown to represent seven new species of Calonectria. These species, provided with the names Ca. eucalypti, Ca. orientalis and Ca. sulawesiensis from Indonesia, Ca. densa, Ca. humicola and Ca. pseudoscoparia from Ecuador and Ca. pini from Colombia were recognised based on morphological characteristics and phylogenetic inference. Recognition of a relatively large number of new species, mainly from soil samples collected in areas not previously intensively sampled, suggests that many more species of Calonectria remain to be discovered, particularly from the tropics and Southern Hemisphere.

Calonectria eucalypti, isolated from the leaves of Eucalyptus grandis, adds a new species to the Ca. colhounii complex (Crous 2002, Crous ), which includes Ca. colhounii, Ca. macroconidialis and Ca. madagascariensis. Members of this complex are characterised by their unique yellow perithecia (Crous 2002). Although Ca. eucalypti was isolated from lesions typical of Cylindrocladium leaf blight, its importance as a pathogen is unknown. Calonectria eucalypti was shown to be homothallic, which is a characteristic that this species shares with Ca. colhounii and Ca. madagascariensis.

The descriptions of Ca. pini and Ca. orientalis add two species to the Ca. brassicae complex (Crous , Lombard ). Calonectria pini was isolated from Pinus patula rooted cuttings with symptoms similar to those associated with root and collar infections caused by Ca. brassicae and Ca. brachiatica on other Pinus spp. (Lombard ). In contrast, Ca. orientalis was isolated from soils collected in Indonesia and nothing is known regarding its pathogenicity. Phylogenetic inference and SNP allele analyses showed that these are closely related sibling species (Taylor ) with genetic isolation having apparently occurred recently. Crosses between isolates of Ca. pini and Ca. orientalis as well as those with themselves and other Calonectria spp. in the group failed to produce perithecia. This is consistent with the observations of Crous et al. (2006) and Lombard et al. (2009), that Calonectria spp. in this complex rarely produce teleomorph structures in culture. Calonectria sulawesiensis resides in the Ca. morganii complex, closely related to Ca. brasiliensis and Ca. insularis. Morphologically, Ca. sulawesiensis can be distinguished from other species in the complex based only on macroconidial dimensions. Therefore phylogenetic inference based on DNA sequence data is necessary to distinguish it from other members of the Ca. morganii complex. Members of this complex are well-known pathogens of various hosts worldwide (Crous 2002), but nothing is known regarding the pathogenicity of Ca. sulawesiensis.

Calonectria pseudoscoparia is a new species in the Ca. scoparia complex (Schoch ), isolated from E. grandis cuttings collected in Ecuador that displayed basal rot symptoms. Calonectria spp. in this group are well known causal agents of cutting rot in commercial forestry nurseries worldwide (Crous , Crous 2002, Lombard ). However, the pathogenicity of Ca. pseudoscoparia is only assumed based on the symptoms with which the fungus was associated.

The two newly described species, Ca. densa and Ca. humicola, isolated from Ecuadorian soils reside in the Ca. spathiphylli complex as defined by Kang et al. (2001b). Calonectria pseudospathiphylli and Ca. spathiphylli, that define this complex, are not easily distinguished based on morphology and DNA sequence comparisons are required for their identification. They can, however, be distinguished based on their mating strategies, with Ca. pseudospathiphylli being homothallic and Ca. spathiphylli being heterothallic (Kang , Crous 2002). The mating strategies of Ca. densa and Ca. humicola could not be determined in this study. This complex of species appears to originate from Central and South America (Chase & Poole 1987, Kang , Crous 2002).

DNA sequence data for the ITS, BT and HIS3 have been used more extensively to explore phylogenetic relationships amongst Calonectria spp. (Schoch , Kang et al. 2001a, 2001b, Henricot & Culham 2002, Crous et al. 2004b, 2006). In this regard, BT is the gene region that provides the most valuable insights into relationships between all species of Calonectria (Schoch et al. 2000b, 2001b, Crous 2002, Henricot & Culham 2002). Application of the CAL and TEF-1α partial gene sequences has only recently been introduced for Calonectria spp. (Crous et al. 2004b, 2006, Lombard et al. 2009, 2010a, c) and data for these gene regions have been available for only a small sub-set of species. The present study has attempted to address this problem and also introduce the ACT and LSU gene sequences that have not been employed previously for Calonectria spp. It has also provided sequence data for all seven gene regions for all accepted species in the genus.

The ITS and LSU sequences provided little valuable information to separate Calonectria spp. In contrast, sequence data for the protein-coding gene regions ACT, BT, CAL, HIS3 and TEF-1α provided good resolution of Calonectria spp., confirming the results of previous studies (Schoch et al. 1999, 2001a, Crous 2002, Henricot & Culham 2002, Crous et al. 2004b, 2006). This study also introduced sequence data for the ACT gene region, although it had few informative sites, consistent with the results of previous studies on other groups of fungi (Helgason , Hunter ). Phylogenetic analyses of the individual coding gene regions and single nucleotide polymorphisms showed that CAL sequence data provide the best resolution distinguishing Calonectria spp. from each other followed by sequence data for the TEF-1α, HIS3, BT and ACT gene regions.

In addition to identifying the most useful gene regions to accurately identify species of Calonectria, an important goal of this study was to re-consider the phylogenetic relationships between all the species in this genus. Having determined that the ACT, BT, CAL, HIS3 and TEF-1α gene regions give the best resolution when identifying species of Calonectria, a phylogenetic tree for the genus was generated. This showed that the group includes two major clades and that these define morphologically similar groups of Calonectria spp. These two major clades have substantial substructure with all of the 66 species of Calonectria residing in one of 13 sub-clades. Eleven of these sub-clades, that include 50 species, represent the Prolate Group of isolates and two sub-clades that include 16 species representing the Sphaero-Naviculate Group of isolates.

The Prolate group of isolates incorporates the majority of the plant pathogenic Calonectria spp. and includes the type species for Calonectria (Ca. pyrochoa) and Cylindrocladium (Cy. scoparium). Most of these pathogenic species have been reported from forestry crops (Peerally 1991, Crous & Wingfield 1994, Crous 2002, Crous ) but a few have also been found to infect horticultural and agronomic crops (Boedijn & Reitsma 1950, Kim , Crous 2002, Polizzi , Vitale et al. 2008). None of the subclades in this group could, however, be correlated with any specific host type.

The geographic distribution of the Calonectria spp. representing the various sub-clades of the unifying Prolate Group of isolates shows some correlation in their distribution. Calonectria spp. in the sub-clade representing the Ca. reteaudii complex (Sub-clade I) have been reported only from Australia, China, Indonesia and New Zealand (Crous 2002, Gadgil & Dick 2004, Crous , Lombard ). Another sub-clade of isolates that appears to have geographical structure resides in the Ca. brassicae complex (Sub-clade IV). Species in this sub-clade, with the exception of Ca. orientalis, have all been reported from South and Central America (Crous 2002, Crous , Lombard ). Isolates in other sub-clades appeared to have broad geographic distribution and not to occur in any defined part of the world.

Species residing in the Sphaero-Naviculate Group had no obvious patterns of pathogenicity, or distribution. This group consisted of two sub-clades in which only vesicle morphology was a consistent character. The majority of the species in the Ca. kyotensis complex (sub-clade XII) have been isolated from debris and soil (Crous ) but a few such as Ca. kyotensis, Ca. ilicicola and Ca. pacifica are important pathogens of agronomic and forestry crops (Crous 2002, Crous ). Members of this sub-clade also had a broad distribution with the majority reported from Asia (Crous ) and they included both heterothallic and homothallic species (Crous 2002, Crous ).

The second sub-clade in the Sphaero-Naviculate Group of isolates (sub-clade XIII) included three Calonectria spp., only two of which have morphological similarities. Calonectria multiphialidica is morphologically similar to the Calonectria spp. in sub-clade XII but there were no obvious patterns of distribution and pathogenicity for this group.

The intention of this phylogenetic study was to include all Calonectria spp. recognised to date. Calonectria curvata and Ca. hederae were, however, not included because there are no cultures for them as has previously been mentioned by Crous (2002). Furthermore, Ca. rajasthanensis, Cy. avesiculatum var. microsporum, Cy. bambusae, Cy. couratarii, Cy. crataegi, Cy. intermedium and Cy. musae were not included due either to the fact that they have not been validly described or not recognised as true species of Calonectria (Crous 2002). Based on the results of this study, 68 Calonectria spp. are recognised as valid and cultures are available for 66 of them.

The teleomorph state has not been seen for several species of Calonectria. Nonetheless Cylindrocladium spp., irrespective of whether their perithecial states are known or not, have been provided names in Calonectria. This is consistent with the view that for all newly described pleomorphic fungal species, the teleomorph name or the oldest typified name takes precedence over the anamorph or more recent name when both types belong to the same holomorph taxon (Hawksworth 2005, McNeill ). It has already been established that Calonectria spp. have only Cylindrocladium anamorphs (Rossman , Schoch ), with micro- and megaconidial states that have thus far not been named. The name Calonectria was typified in 1867 (Rossman 1979) whereas that of Cylindrocladium was typified in 1892 (Morgan 1892). Therefore Calonectria has preference above Cylindrocladium and should henceforth be used for all species irrespective of whether the perithecial state has been found.

KEYS

Both synoptic and dichotomous keys to species of Calonectria are presented. In the synoptic key, numbers grouped with each character refer to the species that are alphabetically arranged below:

Ca. acicola P.D. Gadgill & M.A. Dick

Ca. angustata (Crous & El-Gholl) L. Lombard, M.J. Wingf. & Crous

Ca. asiatica Crous & N.L. Hywel-Jones

Ca. australiensis (Crous & K.D. Hyde) L. Lombard, M.J. Wingf. & Crous

Ca. avesiculata T.S. Schub., El-Gholl, Alfieri & Schoult.

Ca. brachiatica L. Lombard, M.J. Wingf. & Crous

Ca. brassicae (Panwar & Borha) L. Lombard, M.J. Wingf. & Crous

Ca. brasiliensis (Peerally) L. Lombard, M.J. Wingf. & Crous

Ca. canadensis (J.C. Kang, Crous & C.L. Schoch) L. Lombard, M.J. Wingf. & Crous

Ca. cerciana L. Lombard, M.J. Wingf. & Crous

Ca. chinensis (Crous) L. Lombard, M.J. Wingf. & Crous

Ca. citri (H.S. Fawc. & Klotz) L. Lombard, M.J. Wingf. & Crous

Ca. clavata Alfieri, El-Gholl & E.L. Barnard

Ca. colhounii Peerally

Ca. colombiana L. Lombard, M.J. Wingf. & Crous

Ca. colombiensis Crous

Ca. curvata (Boedijn & Reitsma) L. Lombard, M.J. Wingf. & Crous

Ca. curvispora (Crous & D. Victor) L. Lombard, M.J. Wingf. & Crous

Ca. densa L. Lombard, M.J. Wingf. & Crous

Ca. ecuadoriae (Crous & M.J. Wingf.) L. Lombard, M.J. Wingf. & Crous

Ca. eucalypti L. Lombard, M.J. Wingf. & Crous

Ca. gracilipes Crous & G.R.A. Mchau

Ca. gracilis Crous, M.J. Wingf. & Alfenas

Ca. gordoniae (Leahy, T.S. Schub. & El-Gholl) L. Lombard, M.J. Wingf. & Crous

Ca. hawksworthii (Peerally) L. Lombard, M.J. Wingf. & Crous

Ca. hederae C. Booth & J.S. Murray

Ca. hongkongensis Crous

Ca. humicola L. Lombard, M.J. Wingf. & Crous

Ca. hurae (Linder & Whetzel) L. Lombard, M.J. Wingf. & Crous

Ca. ilicicola Boedijn & Reitsma

Ca. indonesiae (Crous) L. Lombard, M.J. Wingf. & Crous

Ca. indusiata (Seaver) Crous

Ca. insularis C.L. Schoch & Crous

Ca. kyotensis Tersh.

Ca. leguminum (Rehm) Crous

Ca. leucothoës (El-Gholl, Leahy & T.S. Schub.) L. Lombard, M.J. Wingf. & Crous

Ca. macroconidialis (Crous, M.J. Wingf. & Alfenas) Crous

Ca. madagascariensis Crous

Ca. malesiana (Crous) L. Lombard, M.J. Wingf. & Crous

Ca. mexicana C.L. Schoch & Crous

Ca. morganii Crous, Alfenas & M.J. Wingf.

Ca. multiphialidica (Crous, Simoneau & Risède) L. Lombard, M.J. Wingf. & Crous

Ca. multiseptata Crous & M.J. Wingf.

Ca. naviculata Crous & M.J. Wingf.

Ca. orientalis L. Lombard, M.J. Wingf. & Crous

Ca. ovata D. Victor & Crous

Ca. pacifica (J.C. Kang, Crous & C.L. Schoch) L. Lombard, M.J. Wingf. & Crous

Ca. pauciramosa C.L. Schoch & Crous

Ca. penicilliodes (Tubaki) L. Lombard, M.J. Wingf. & Crous

Ca. pini L. Lombard, M.J. Wingf. & Crous

Ca. polizzii L. Lombard, M.J. Wingf. & Crous

Ca. pseudonaviculata (Crous, J.Z. Groenew. & C.F. Hill) L. Lombard, M.J. Wingf. & Crous

Ca. pseudoreteaudii L. Lombard, M.J. Wingf. & Crous

Ca pseudoscoparia L. Lombard, M.J. Wingf. & Crous

Ca. pseudospathiphylli J.C. Kang, Crous & C.L. Schoch

Ca. pteridis Crous, M.J. Wingf. & Alfenas

Ca. pyrochoa (Desm.) Sacc.

Ca. queenslandica L. Lombard, M.J. Wingf. & Crous

Ca. reteaudii (Bugn.) C. Booth

Ca. rumohrae El-Gholl & Alfenas

Ca. scoparia Peerally

Ca. spathiphylli El-Gholl, J.Y. Uchida, Alfenas, T.S. Schub., Alfieri & A.R. Chase

Ca. spathulata El-Gholl, Kimbr., E.L. Barnard, Alfieri & Schoult.

Ca. sulawesiensis L. Lombard, M.J. Wingf. & Crous

Ca. sumatrensis (Crous) L. Lombard, M.J. Wingf. & Crous

Ca. terrae-reginae L. Lombard, M.J. Wingf. & Crous

Ca. variabilis Crous, B.J.H. Janse, D. Victor, G.F. Marias & Alfenas

Ca. zuluensis L. Lombard, M.J. Wingf. & Crous

Synoptic key to Calonectria species

Teleomorph:

Teleomorph state known

1, 3, 5, 13, 14, 15, 16, 21, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46, 48, 55, 56, 57, 59, 60, 61, 62, 63, 67, 68

Teleomorph state unknown

2, 4, 6, 7, 8, 9, 10, 11, 12, 17, 18, 19, 20, 24, 25, 28, 36, 39, 42, 45, 47, 49, 50, 51, 52, 53, 54, 58, 64, 65, 66

Ascocarps:

Red-brown to red in colour, changing to dark-red in 3 % KOH

1, 23, 44, 56, 61, 67

Orange to red in colour, changing to dark-red in 3 % KOH

3, 5, 15, 16, 22, 26, 30, 32, 33, 34, 40, 43, 55, 62, 68

Orange to red-brown in colour, changing to dark-red in 3 % KOH

13, 27, 35, 46, 48, 57, 59, 60, 63

Yellow to orange in colour, only base and stroma changing to dark-red in 3 % KOH

14, 21, 37, 38, 41

Asci:

8-spored and clavate

1, 3, 5, 13, 15, 16, 22, 23, 26, 27, 30, 32, 33, 34, 35, 38, 40, 41, 43, 44, 46, 48, 55, 56, 57, 59, 60, 61, 62, 63, 67, 68

4-spored and clavate

14, 21, 37

Ascospore septation:

1-septate

3, 15, 16, 22, 23, 27, 33, 34, 40, 41, 48, 61, 68

(1–)3-septate

5, 13, 14, 21, 26, 30, 32, 35, 37, 38, 44, 46, 55, 56, 57, 59, 62, 63, 67

(3–)4-septate

1

(1–)3–6(–9) septate

43, 60

Ascospore width (av. in μm)

4–5

15, 16, 22, 34, 44, 62, 67, 68

5.5–6

1, 3, 5, 13, 14, 21, 26, 27, 30, 33, 37, 38, 40, 41, 46, 55, 56, 57, 59, 61, 63

6.5–7

22, 32, 35, 43, 48, 60

Ascospore length (av. in μm)

30–39

3, 15, 16, 21, 22, 23, 27, 33, 34, 41, 48, 68

40–49

5, 13, 30, 44, 55, 57, 61, 62, 67

50–59

14, 26, 32, 37, 38, 40, 56, 63

60–69

46

70 and above

1, 35, 43, 59, 60

Stipe length (av. in μm)

40–100

1, 5, 6, 9, 10, 16, 18, 20, 21, 27, 30, 31, 33, 34, 36, 38, 40, 44, 47, 48, 49, 50, 57, 58, 61, 63, 65, 66, 68

101–150

4, 7, 11, 13, 15, 24, 32, 41, 42, 51, 53, 54, 60, 62, 64,

151–200

2, 3, 12, 14, 19, 22, 23, 28, 29, 35, 39, 45, 46, 52, 56, 67

above 200

25, 26, 37, 55, 59

Stipe extension length (av. in μm)

Less than 100

1

100–200

9, 11, 12, 15, 16, 18, 19, 25, 27, 28, 31, 34, 39, 41, 44, 51, 52, 57, 58, 68

201–300

2, 3, 10, 13, 14, 21, 22, 24, 26, 30, 33, 35, 36, 40, 45, 46, 47, 48, 50, 54, 55, 56, 61, 62, 63, 64, 65, 66, 67

Above 300

4, 5, 6, 7, 20, 23, 29, 32, 37, 38, 42, 53, 59, 60

Vesicle shape

Avesiculate to clavate

5

Clavate

1, 2, 4, 6, 7, 13, 14, 20, 21, 22, 23, 24, 29, 32, 35, 37, 38, 43, 45, 50, 53, 56, 58, 59, 60, 64, 66

Ellipsoidal to pyriform to obovoid

8, 12, 25, 26, 41, 55, 61, 63

Ellipsoidal to ovoid

19, 46

Ellipsoidal to obpyriform

10, 15, 33, 36, 40, 48, 51, 54, 57, 68

Sphaeropedunculate

3, 9, 11, 16, 17, 18, 19, 27, 30, 31, 34, 39, 42, 47, (49), 64, 67

Globose

19, 28, 62

Naviculate

44, 52

Shape of phialides on macroconidiophore

Reniform to doliiform

3, 6, 7, 8, 9, 10, 12, 15, 17, 19, 20, 21, 22, 23, 24, 25, 26, 33, 34, 36, 40, 41, 44, 45, 46, 48, 49, 50, 51, 52, 54, 57, 61, 63, 64, 68

Elongate reniform to doliiform

5, 11, 13, 14, 16, 18, 27, 28, 30, 31, 39, 42, 47, 55, 56, 62, 65, 67

Cylindrical to allantoid

1, 2, 4, 29, 32, 35, 37, 38, 53, 58, 59, 60, 66

Number of fertile branches on macroconidiophore

1–3

1, 5, 8, 9, 11, 12, 17, 18, 28, 30, 46, 48, 49, 50, 51, 52, 53, 57, 58, 60, 63, 66, 67, 68

4–6

2, 3, 4, 6, 7, 14, 16, 19, 21, 24, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 44, 45, 46, 47, 54, 55, 56, 59, 61, 62, 64, 65

More than 6

20, 27, 42

Microconidia

Microconidia absent

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 47, 48, 49, 50, 51, 52, 54, 55, 57, 58, 61, 63, 64, 65, 66, 68

Microconidia present

1, 13, 24, 29, 30, 43, 46, 53, 56, 59, 60, 62, 67

Microconidial septation

1-septate

13, 29, 30, 46, 56, 62, 67

1(–3)-septate

24, 59, 60

1–3-septate

1, 43, 53

Microconidial width (mean in μm)

Up to 3

13, 29, 43, 46, 56, 59

Up to 4

24, 53, 62, 67

Up to 5

1, 30, 60

Microconidial length (mean in μm)

Below 20

29

20–30

1, 30, 46, 56, 59, 60, 67

31–40

13, 24, 62

above 40

43, 53

Macroconidial septation

1-septate

3, 6, 7, 8, 9, 10, 11, 12, 15, 17, 19, 22, 25, 27, 28, 31, 33, 34, 39, 40, 41, 42, 44, 45, 47, 48, 50, 51, 52, 54, 61, 64, 65, 68

1(–3)-septate

5, 13, 16, 18, 20, 23, 24, 36, 46, 53, 55, 56, 62

(1–)3-septate

4, 14, 21, 30, 32, 38, 49, 57,

(1–)3(–6)-septate

26, 37, 58, 66

(1–)5(–6)-septate

1, 26, 35, 59, 60

(1–)7(–8)-septate

29

More than 8-septate

2

Macroconidial width (av. in μm)

3–4

8, 9, 11, 12, 15, 17, 25, 27, 31, 33, 34, 39, 40, 41, 44, 45, 51, 54, 55, 63, 64, 68

4.5–5

3, 5, 6, 7, 10, 13, 14, 16, 18, 20, 22, 23, 24, 28, 35, 36, 38, 42, 46, 47, 48, 49, 50, 52, 61, 65, 67

5.5–6

19, 21, 26, 30, 32, 56, 57, 58, 62, 66

6.5–7

1, 4, 37, 59

above 7

2, 29, 53, 60

Macroconidial length (av. in μm)

Less than 40

8, 15, 51, 68

40–46

6, 10, 11, 17, 22, 30, 33, 34, 40, 41, 44, 50

47–55

3, 7, 9, 14, 16, 19, 20, 27, 28, 31, 38, 39, 42, 45, 47, 48, 49, 52, 54, 55, 63, 64

56–66

4, 5, 12, 13, 18, 23, 24, 25, 26, 35, 57, 61, 65

67–75

1, 21, 36, 46, 58, 62, 67

76–95

32, 37, 56, 59, 66

above 95

29, 53, 60

Dichotomous key to Calonectria species

The following key is an adaptation of the key provided by Crous (2002) to include all Calonectria spp. described subsequent to 2002. Measurements and observations are those of Crous (2002) and other authors who have described species subsequent to 2002 (Table 1). Only average conidial dimensions, where available, and a few distinguishing characters are presented in the key. Complete descriptions should be consulted to determine species variations. Calonectria penicilloides has been omitted from the keys, due to the fact that there is little morphological information available for this species.

1. Stipe extensions thick-walled; acicular to clavate vesicles................................................................................................................ 2 1. Stipe extensions and vesicles not as above.................................................................................................................................... 28

2. Stipe extensions thick-walled, terminating in acicular to clavate vesicles; fertile branches –3; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 64 × 5 μm; perithecia orange to red; ascospores 1(–3)-septate, 40 × 6 μm......................................................................................................................... 2. Stipe extensions not thick-walled and vesicles clavate...................................................................................................................... 3

3. Teleomorph state unknown................................................................................................................................................................ 4 3. Teleomorph state known.................................................................................................................................................................. 15

4. Macroconidia 1-septate only.............................................................................................................................................................. 5 4. Macroconidia more than 1-septate..................................................................................................................................................... 8

5. Fertile branches –3; phialides doliiform to reniform; macroconidia 1-septate, 44 × 5 μm; stipe extensions terminating in clavate vesicles...................................................................................................................... 5. Fertile branches –5............................................................................................................................................................................ 6

6. Lateral stipe extensions present; macroconidia 1(–2)-septate, 44 × 5 μm; stipe extensions terminating in clavate vesicles; fertile branches –5; phialides doliiform to reniform....................................................................... 6. Lateral stipe extensions absent.......................................................................................................................................................... 7

7. Stipe extensions terminating in clavate vesicles; fertile branches –5; phialides doliiform to reniform; macroconidia 1-septate, 53 × 4.5 μm............................................................................................................................ 7. Stipe extensions terminating in clavate vesicles; fertile branches –5; phialides doliiform to reniform; macroconidia 1-septate, 48 × 4 μm...............................................................................................................................

8. Macroconidia longer than 100 μm...................................................................................................................................................... 9 8. Macroconidia shorter than 100 μm................................................................................................................................................... 10

9. Macroconidia 5–8-septate, 104 × 8 μm; stipe extension terminate in clavate vesicles; fertile branches –3; phialides cylindrical to allantoid; microconidiophores lacking stipe extension; microconidia 1–3-septate, 44 × 4 μm................................................................................................................ 9. Macroconidia 1–3-septate................................................................................................................................................................. 12

10. Macroconidia (1–)3-septate, 63 × 6.5 μm; stipe extensions terminating in clavate vesicles; fertile branches –6; phialides cylindrical to allantoid................................................................................................ 10. Macroconidia 1(–3)-septate.............................................................................................................................................................. 11

11. Fertile branches –7; phialides doliiform to reniform; macroconidia 51 × 4.5 μm; stipe extensions terminating in clavate vesicles.......................................................................................................... 11. Fertile branches –4; phialides doliiform to reniform; macroconidia 62 × 5 μm; stipe extensions terminating in clavate vesicles............................................................................................................

12. Macroconidia longer than 100 μm with more than 6 septa............................................................................................................... 13 12. Macroconidia shorter than 100 μm with 6 or less septa................................................................................................................... 14

13. Stipe extensions terminating in narrowly clavate vesicles; fertile branches –4; phialides cylindrical; macroconidia (1–)7–10(–12)-septate with slight swelling in the middle, 110 × 10 μm; Mega- and microconidia absent.................................................................................................................................... 13. Stipe extensions terminating in narrowly clavate vesicles; fertile branches –3; phialides cylindrical; microconidia present, 1-septate, 18 × 3 μm; macroconidia (1–)7(–8)-septate, 120 × 7.5 μm; megaconidia present, 9–16-septate, bent or curved, (150–)200–250(–270) × 6–7(–8) μm................................................

14. Stipe extensions terminating in narrowly clavate vesicles; fertile branches –3; phialides cylindrical to allantoid, obpyriform when carried singly; macroconidia 4–6-septate, 69 × 6 μm............................................................... 14. Stipe extensions terminating in a narrowly clavate vesicles; fertile branches –3; phialides cylindrical to allantoid, obpyriform when carried singly; macroconidia 4–6-septate, 76 × 6 μm................................................................

15. Macroconidial state unknown; megaconidiophores with stipe extensions terminating in clavate vesicles when present; megaconidia 6–10-septate, boomerang-shaped or curved, (120–)150–170(–220) × 8–9 μm; microconidia 1–3-septate, straight or curved, 20–65 × 2.5–3.5 μm.......................................................................... 15. Macroconidial state known................................................................................................................................................................ 16

16. Teleomorph state known and macroconidia 1-septate to 1(–3)-septate........................................................................................... 17 16. Teleomorph state known and macroconidia multi-septate................................................................................................................ 20

17. Teleomorph homothallic.................................................................................................................................................................... 18 17. Teleomorph heterothallic................................................................................................................................................................... 19

18. Perithecia orange with a red apex; ascospores 1-septate, 35 × 6.5 μm; stipe extensions terminating in clavate vesicles; fertile branches –4; phialides doliiform to reniform; macroconidia 1-septate, 45 × 4.5 μm........................................... 18. Perithecia red; ascospores 1-septate, 37 × 5 μm; stipe extensions terminating in narrowly clavate vesicles; fertile branches –4; phialides doliiform to reniform; macroconidia 1(–3)-septate, 56 × 4.5 μm.........................................

19. Perithecia orange; ascospores 1(–3)-septate, 44 × 5.5 μm; stipe extensions terminating in narrowly clavate vesicles; fertile branches –4; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 65 × 5 μm; microconidia 1-septate, 32 × 3 μm.................................................................................................................................... 19. Perithecia red-brown; ascospores 1(–3)-septate, 52 × 6 μm; stipe extensions terminating in clavate to narrowly ellipsoidal vesicles; fertile branches –5; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 82 × 5.5 μm; microconidia 1-septate, 30 × 3.5 μm.............................................................

20. Macroconidia 3-septate..................................................................................................................................................................... 21 20. Macroconidia 3- to multi-septate....................................................................................................................................................... 25

21. Perithecia yellow to orange............................................................................................................................................................... 22 21. Perithecia yellow............................................................................................................................................................................... 23

22. Teleomorph state homothallic; perithecia yellow to orange; ascospores (1–)3-septate, 33 × 6 μm; stipe extensions terminating in broadly clavate vesicles; fertile branches –5; phialides doliiform to reniform; macroconidia 3-septate, 72 × 6 μm............................................................................... 22. Teleomorph state homothallic; perithecia orange to red; ascospores (1–)3-septate, 53 × 7 μm; stipe extensions terminating in narrowly clavate vesicles; fertile branches –5; phialides allantoid to reniform; macroconidia (1–)3-septate, 81 × 6 μm; megaconidia 7–9(–14)-septate, boomerang-shaped to curved, 130–200 × 5–6 μm........................................................................................................

23. Macroconidia and ascospores shorter than 65 μm; teleomorph state homothallic; perithecia bright yellow; ascospores (1–)3-septate, 50 × 5.5 μm; stipe extensions terminating in clavate vesicles; fertile branches –5; phialides allantoid to cylindrical; macroconidia (1–)3-septate, 55 × 4.5 μm.................... 23. Macroconidia and ascospores longer than 65 μm............................................................................................................................ 24

24. Teleomorph state homothallic; perithecia bright yellow; ascospores (1–)3-septate, 55 × 6 μm; stipe extensions terminating in clavate vesicles; fertile branches –5; phialides elongate-doliiform to reniform; macroconidia (1–)3-septate, 65 × 5 μm......................................................... 24. Teleomorph state heterothallic; perithecia dirty yellow, ascospores (1–)3-septate, 55 × 6 μm; stipe extensions terminating in clavate vesicles; fertile branches –5; phialides allantoid to cylindrical; macroconidia (1–)3(–4)-septate, 90 × 6.5 μm....................................................................................................

25. Macroconidiophore branches –2 or less........................................................................................................................................... 26 25. Macroconidiophore with more than 2 series of branches.................................................................................................................. 27

26. Teleomorph state homothallic; perithecia orange-brown; ascospores 3–6(–9)-septate, 90 × 6.5 μm; stipe extensions terminating in narrowly clavate vesicles; fertile branches –2; phialides cylindrical; microconidia 1(–3)-septate, (8–)15–30(–50) × 3–5 μm; macroconidia 5(–7)-septate, 110 × 9 μm; megaconidia 7–13-septate, bent or curved, (120–)180–230 × (8–)10–11(–13) μm...................................................... 26. Teleomorph state homothallic; perithecia red to red-brown; ascospores 3–4-septate, 70 × 6 μm; stipe extensions, when present, terminating in narrowly clavate vesicles; fertile branches –1; macroconidia 5–7-septate, 75 × 7 μm; microconidia 1–3-septate, 10–30 × 3–5 μm........................................................

27. Teleomorph state homothallic; perithecia orange to red-brown; ascospores (1–)3-septate, 70 × 6.5 μm; stipe extensions terminating in narrowly clavate vesicles; fertile branches –6; phialides cylindrical to allantoid; macroconidia (1–)3–5(–6)-septate, 60 × 5 μm........................................................................................................... 27. Teleomorph state heterothallic; perithecia orange to red-brown; ascospores (1–)5(–6)-septate, 70 × 5.5 μm; stipe extensions terminating in clavate vesicles; fertile branches –6; phialides cylindrical to allantoid; macroconidia (1–)5(–6)-septate, 84 × 6.5 μm; microconidia 1(–3)-septate, 30 × 3 μm.................................................

28. Vesicles sphaeropedunculate, globose or ovoid............................................................................................................................... 29 28. Vesicles not as above....................................................................................................................................................................... 48

29. Vesicles consistently ovate; teleomorph state heterothallic; perithecia orange; ascocpores 1–3(–7)-septate, 60 × 5.5 μm; fertile branches –3; phialides doliiform to reniform; macroconidia straight or curved, 1(–3)-septate, 70 × 5 μm; microconidia 1-septate, 21 × 3 μm............................................................................................. 29. Vesicles not consistently ovate......................................................................................................................................................... 30

30. Macroconidia 1(–3)-septate.............................................................................................................................................................. 31 30. Macroconidia only 1-septate............................................................................................................................................................. 36

31. Teleomorph state unknown; stipe extensions terminating in sphaeropedunculate vesicles; fertile branches –3; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 60 × 5 μm....................................................... 31. Teleomorph state known................................................................................................................................................................... 32

32. Perithecia red-brown; teleomorph state homothallic; ascospores 1(–3)-septate, 42 × 5 μm; stipe extensions terminating in sphaeropedunculate to ovoid or ellipsoidal to clavate vesicles; fertile branches –3; phialides elongate-doliiform to reniform; macroconidia (1–)3(–4)-septate, 73 × 5 μm; microconidia 1-septate, 27 × 4 μm................................................................................................................................. 32. Perithecia orange to red.................................................................................................................................................................... 33

33. Teleomorph state heterothallic; perithecia orange to red; ascospores 1(–3)-septate, 45 × 5 μm; stipe extensions terminating in globoid or ellipsoid to obpyriform vesicles; fertile branches –5; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 70 × 6 μm; microconidia 1-septate, 39 × 4 μm............................................................................................................................ 33. Teleomorph state homothallic........................................................................................................................................................... 34

34. Lateral stipe extensions abundant; perithecia orange; ascospores 1-septate, 33 × 5 μm; stipe extensions terminating in sphaeropedunculate vesicles; fertile branches –5; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 53 × 4.5 μm............................................... 34. Lateral stipe extensions absent......................................................................................................................................................... 35

35. Ascospores 1(–3)-septate, 42 × 5.5 μm; stipe extensions terminating in sphaeropedunculate to ellipsoidal vesicles; fertile branches –4; phialides elongate-doliiform to reniform; macroconidia 1(–3)-septate, 52 × 4 μm........ 35. Ascospores 1(–3)-septate, 45 × 6 μm; stipe extensions terminating in sphaeropedunculate vesicles; fertile branches –3; phialides elongate-doliiform to reniform; macroconidia (1–)3-septate, 62 × 6 μm; microconidia 1-septate, 30 × 4.5 μm................................................................................................................................

36. Stipe thick-walled; teleomorph state unknown; stipe extensions terminating in clavate to sphaeropedunculate vesicles; fertile branches –8; phialides elongate-doliiform to reniform; macroconidia 1-septate, 53 × 4.5 μm.................... 36. Stipe thin-walled................................................................................................................................................................................ 37

37. Teleomorph state known................................................................................................................................................................... 38 37. Teleomorph state unknown............................................................................................................................................................... 40

38. Macroconidiophore branches –8; perithecia orange; teleomorph state homothallic; perithecia orange; ascospores 1-septate, 31 × 6 μm; stipe extensions terminating in sphaeropedunculate vesicles; phialides elongate-doliiform to reniform; macroconidia 1-septate, 46.5 × 4 μm.................................................. 38. Macroconidiophore branches –5....................................................................................................................................................... 39

39. Teleomorph state homothallic; perithecia orange; ascospores 1-septate, 33 × 6 μm; stipe extensions terminating in sphaeropedunculate vesicles, lateral stipe extensions abundant; phialides doliiform to reniform; macroconidia 1-septate, 53 × 5 μm................................................................................. 39. Teleomorph state homothallic; perithecia orange to red; ascospores 1-septate, 35 × 5 μm; stipe extensions terminating in sphaeropedunculate vesicles, lateral stipe extensions abundant; phialides doliiform to reniform; macroconidia 1-septate, 40 × 3.5 μm..........................................................................

40. Lateral stipe extensions absent......................................................................................................................................................... 41 40. Lateral stipe extensions present....................................................................................................................................................... 43

41. Macroconidia curved, 1-septate, 40–46 × 3–4 μm; stipe extensions terminating in sphaeropedunculate vesicles; fertile branches –2............................................................................................................................................................. 41. Macroconidia straight........................................................................................................................................................................ 42

42. Stipe extensions terminating in globose to ovoid to sphaeropedunculate vesicles; fertile branches –3; phialides doliiform to reniform; macroconidia 1-septate, 51 × 5 μm............................................................................. 42. Stipe extensions terminating in sphaeropedunculate vesicles; fertile branches –5; phialides elongate-doliiform to reniform; macroconidia 1-septate, 50.5 × 4 μm..........................................................

43. Lateral stipe extensions rare; stipe extensions terminating in pyriform to sphaeropedunculate vesicles; fertile branches – 3; phialides doliiform to reniform; macroconidia 1-septate, 50 × 4 μm........................................... 43. Lateral stipe extensions abundant.................................................................................................................................................... 44

44. Macroconidiophore branches 4–6..................................................................................................................................................... 45 44. Macroconidiophore branches –3....................................................................................................................................................... 46

45. Macroconidiophore branches –4; stipe extension terminating in globose to ovoid to sphaeropendunculate vesicles; phialides doliiform to reniform; macroconidia 1-septate, 54 × 6 μm.................................................................................... 45. Macroconidiophore branches –6; stipe extensions terminating in sphaerpendunculate vesicles; phialides elongate-doliiform to reniform; macroconidia 1-septate, 47.5 × 4 μm...........................................................

46. Macroconidia 45 × 4 μm, 1-septate; stipe extensions terminating in sphaeropedunculate vesicles; phialides elongate-doliiform to reniform......................................................................................................................... 46. Macroconidia longer than 45 μm...................................................................................................................................................... 47

47. Stipe extensions terminating in sphaeropedunculate vesicles; phialides elongate-doliiform to reniform; macroconidia 1-septate, 55 × 4.5 μm............................................................................................................................... 47. Stipe extensions terminating in sphaeropedunculate vesicles; phialides elongate-doliiform to reniform; macroconidia 1-septate, 58 × 5 μm..........................................................................................................................

48. Vesicles pyriform to ellipsoidal or clavate, rarely ovoid, never obpyriform........................................................................................ 49 48. Vesicles not as above....................................................................................................................................................................... 54

49. Macroconidia more than 1-septate.................................................................................................................................................... 50 49. Macroconidia 1-septate..................................................................................................................................................................... 51

50. Teleomorph state unknown; stipe extensions terminating in narrowly ellipsoidal to pyriform or ovoid to sphaeropedunculate vesicles; fertile branches –3; phialides doliiform to reniform; macroconidia (1–)3-septate, 58 × 4 μm................................................................................................................................... 50. Teleomorph state homothallic; perithecia orange-red; ascospores 1(–3)-septate, 33.5–69 × 4.5–7 μm; stipe extensions terminating in clavate to ovoid or ellipsoidal vesicles; fertile branches –4; phialides doliiform to reniform; macroconidia (1–)3(–5)-septate, (44–)50–70(–102) × 5–7(–8) μm................................

51. Stipe extensions up to 200 μm long................................................................................................................................................. 52 51. Stipe extensions longer than 200 μm............................................................................................................................................... 53

52. Teleomorph state heterothallic; perithecia yellow to orange; ascospores 1-septate, 37 × 6 μm; stipe extensions terminating in ellipsoidal to pyriform or clavate vesicles; fertile branches –6; phialides doliiform to reniform; macroconidia 1-septate, 45 × 4 μm................................................................................ 52. Teleomorph state unknown; stipe extensions terminating in oval to ellipsoidal to fusiform vesicles; fertile branches –3; phialides doliiform to reniform; macroconidia 1-septate, 38 × 3.5 μm........................................

53. Macroconidia curved, 1-septate, 56 × 4 μm, stipe extensions terminating in ellipsoidal to clavate vesicles; fertile branches –4; phialides doliiform to reniform; teleomorph state unknown..................................................... 53. Macroconidia straight, 1-septate, 48 × 4 μm Teleomorph state unknown; stipe extensions terminating in broadly clavate to ellipsoidal vesicles; fertile branches –5; phialides doliiform to reniform;...................................

54. Vesicles obpyriform to ellipsoidal...................................................................................................................................................... 55 54. Vesicles naviculate............................................................................................................................................................................ 66

55. Macroconidia 1-septate..................................................................................................................................................................... 56 55. Macroconidia more than 1-septate.................................................................................................................................................... 64

56. Macroconidiophore branches –3....................................................................................................................................................... 57 56. Macroconidiophore branches 4–6..................................................................................................................................................... 59

57. Teleomorph state homothallic; perithecia orange to red; ascospores 1-septate, 32 × 4 μm; stipe extensions terminating in broadly clavate to obpyriform vesicles; phialides doliiform to reniform; macroconidia 1-septate, 36 × 4 μm............................................................................................................................... 57. Teleomorph state heterothallic.......................................................................................................................................................... 58

58. Perithecia orange to red-brown; ascospores 1-septate, 35 × 6.5 μm; stipe extensions terminating in obpyriform to ellipsoidal vesicles; phialides doliiform to reniform; macroconidia 1-septate, 50 × 4.5 μm................ 58. Teleomorph state unknown; stipe extensions terminating in broadly clavate to obpyriform vesicles; phialides doliiform to reniform; macroconidia 1-septate, 37 × 4 μm...................................................................................

59. Macroconidia up to 45 μm long........................................................................................................................................................ 60 59. Macroconidia longer than 45 μm...................................................................................................................................................... 63

60. Macroconidiophore branches –6; teleomorph state heterothallic; perithecia orange to red; ascospores 1-septate, 33 × 6 μm; stipe extensions terminating in obpyriform to broadly ellipsoidal vesicles; phialides doliiform to reniform; macroconidia 1-septate, 45 × 4 μm................................................................................ 60. Macroconidiophore branches –4....................................................................................................................................................... 61

61. Vesicles broadly ellipsoidal with a papillate apex; phialides doliiform to reniform; macroconidia 1-septate, 45 × 4 μm; teleomorph state heterothallic; perithecia orange to red; ascospores 1-septate, 50 × 5.5 μm...................................... 61. Vesicles fusiform to obpyriform......................................................................................................................................................... 62

62. Teleomorph state homothallic; perithecia yellow to orange; ascospores 1-septate, 34 × 4 μm; phialides doliiform to reniform; macroconidia 1-septate, 37 × 3 μm.......................................................................... 62. Teleomorph state unknown; phialides doliiform to reniform; macroconidia 1-septate, 44 × 5 μm...................................

63. Teleomorph state heterothallic; perithecia red-brown; ascospores 1-septate, 48 × 5.5 μm; stipe extensions terminating in ellipsoidal to narrowly obpyriform vesicles; fertile branches –5; phialides doliiform to reniform; macroconidia 1-septate, 60 × 4.5 μm............................................................................ 63. Teleomorph state unknown; stipe extensions terminating in obpyriform to ellipsoidal vesicles; fertile branches –4; phialides doliiform to reniform; macroconidia 1-septate, 48 × 4 μm....................................

64. Macroconidiophore branches –6; stipe extensions terminating in ellipsoidal to obpyriform vesicles; phialides cylindrical, straight or doliiform to reniform; macroconidia 1(–3)-septate, 73 × 5 μm.................................. 64. Macroconidiophore branches –3....................................................................................................................................................... 65

65. Teleomorph state homothallic; perithecia orange to red-brown; ascospores 1(–3)-septate, 50 × 5.5 μm; stipe extensions terminating in obpyriform to broadly ellipsoidal vesicles; phialides doliiform to reniform; macroconidia (1–)3-septate, 50–70 × 5–6 μm............................................................................................................... 65. Teleomorph state homothallic; perithecia orange; ascospores (1–)3-septate, 50 × 5.5 μm; stipe extensions terminating in ellipsoidal to obpyriform or clavate vesicles; phialides cylindrical, straight or doliiform to reniform; macroconidia (1–)3(–6)-septate, 55 × 4 μm..............................................................

66. Teleomorph state heterothallic; perithecia red-brown; ascospores 1(–3)-septate, 40 × 5 μm; fertile branches –5; phialides doliiform to reniform; macroconidia 1-septate, 45 × 3 μm.............................................. 66. Teleomorph state unknown; fertile branches –3; phialides doliiform to reniform; macroconidia 1-septate, 42–68 × 4–6 μm.......................................................................................................