Biodiversity of Lecanosticta pine-needle blight pathogens suggests a Mesoamerican Centre of origin.

Lecanosticta acicola causes the disease known as brown spot needle blight (BSNB), on Pinus species. The pathogen is thought to have a Central American centre of origin. This was based on the morphological variation between isolates believed to represent L. acicola from native Pinus spp. Two species of Lecanosticta, L. brevispora and L. guatemalensis, have recently been described from Mexico and Guatemala respectively based on morphology and sequence-derived phylogenetic inference. However, the putative native pathogen, L. acicola, was not found in those areas. In this study, the species diversity of a large collection of Lecanosticta isolates from Central America was considered. Phylogenetic analyses of the BT1, ITS, MS204, RPB2 and TEF1 gene regions revealed six species of Lecanosticta, four of which represented undescribed taxa. These are described here as Lecanosticta jani sp. nov. from Guatemala and Nicaragua, L. pharomachri sp. nov. from Guatemala and Honduras, L. tecunumanii sp. nov. from Guatemala and L. variabilis sp. nov. from Guatemala, Honduras, and Mexico. New host and country records were also found for the previously described L. brevispora and L. guatemalensis. Lecanosticta acicola was not found in any of the samples from Central America, and we hypothesize that it could be a northern hemisphere taxon. The high species diversity of Lecanosticta found in Mesoamerica suggests that this is a centre of diversity for the genus.

INTRODUCTION

Brown spot needle blight (BSNB) or Lecanosticta needle blight is an important needle disease on Pinus species. The disease is characterised by brown spots on necrotic yellow lesions at the points of infection and die-back of the needles from the apex, which often leads to premature defoliation (Ivory 1987). BSNB is caused by the fungal pathogen, Lecanosticta acicola (Siggers 1944). The fungus is a well-known pathogen in the USA and has also been recorded in Central America, Colombia, Europe as well as Asian countries including China, Japan and Korea. Lecanosticta acicola is regarded as an A2 quarantine pathogen in Europe and Colombia where it is present as well as an A1 quarantine pathogen in the rest of South America (COSAVE), Africa (IASPC) and the Eurasian Economic Union countries where it has yet to be recorded (https://gd.eppo.int/taxon/SCIRAC/categorization). Despite its quarantine status, L. acicola has been discovered in various new locations and on new hosts in Europe during the past decade (Jankovsky et al. 2009; Markovskaja et al. 2011; Anonymous 2012; Hintsteiner et al. 2012; Adamson et al. 2015; Janoušek et al. 2016; Ortíz de Urbina et al. 2017; Mullett et al. 2018; Cleary et al. 2019; Sadiković et al. 2019).

Siggers (1944) and Evans (1984) summarised the taxonomic and nomenclatural history of Lecanosticta acicola, which was complicated by the former system which allowed asexual and sexual morphs of the same species of fungi to be given separate scientific names (Kais 1971; Evans 1984). From 1972 to 2012, the name Mycosphaerella dearnessii was widely used for the causal agent of BSNB. It was, however, recently recognised that Mycosphaerella is polyphyletic and should be strictly used for fungi in Ramularia (Crous et al. 2007; Crous 2009). Following the One Fungus One Name (1F1N) convention (Hawksworth et al. 2011), the nomenclatural rules were changed in July 2011, and included in subsequent editions of the International Code of Nomenclature for algae, fungi, and plants (ICN) (Turland et al. 2018). Lecanosticta was taken up as the appropriate name, with L. acicola as type species of the genus (Crous et al. 2009a; Quaedvlieg et al. 2012).

Five species of Lecanosticta have been described: Lecanosticta acicola, L. brevispora, L. guatemalensis (Quaedvlieg et al. 2012), L. gloeospora (Evans 1984), and L. longispora (Marmolejo 2000). Lecanosticta acicola remains the best-known species and records suggest that it has a wide distribution in North and South America, Europe, and Asia (https://gd.eppo.int/taxon/SCIRAC/distribution). The remaining four species are known only from Mesoamerica (Evans 1984; Marmolejo 2000; Quaedvlieg et al. 2012). Lecanosticta gloeospora was described, based only on morphology, from disease symptoms on Pinus pseudostrobus from Iturbide, Nuevo León, Mexico (Evans 1984). It was subsequently reported on P. pseudostrobus collected in 1990 in Mexico (Marmolejo 2000). Lecanosticta longispora was originally described from Pinus culminicola in Nuevo León, Mexico, based on morphology (Marmolejo 2000). Quaedvlieg et al. (2012) redescribed and epitipified L. longispora based on DNA sequence and morphological data. Quaedvlieg et al. (2012) delineated Mycosphaerella species of quarantine significance in Europe, including isolates believed to be L. acicola from Central America. Those isolates were distinct taxa and were named L. brevispora and L. guatemalensis from Pinus sp. in Mexico and from P. oocarpa in Guatemala.

Names assigned to Lecanosticta species prior to 2012 were based only on morphological characteristics. Cryptic diversity in Lecanosticta is illustrated by L. guatemalensis (IMI281598), which was initially identified as L. acicola (Evans 1984; Quaedvlieg et al. 2012). Identifications made utilising only morphological characteristics should clearly be re-evaluated using DNA sequence data and phylogenetic inference.

Central America is believed to be the centre of origin of L. acicola. This hypothesis was first proposed by Evans (1984), when the fungus was isolated from native trees in pristine forests. In a recent phylogenetic study, high levels of diversity were found in the Translation Elongation 1-α gene region (TEF1) of isolates from Mexico and Guatemala (Janoušek et al. 2016). Furthermore, Central American isolates did not group in the same clade as isolates from Asia, Europe, and North America. Likewise, Janoušek et al. (2016) reported poor amplification of microsatellite regions that had been developed for L. acicola suggesting that the isolates could represent cryptic species. The present study emerged from an opportunity to collect pine needles infected with Lecanosticta spp. in Guatemala, Honduras and Nicaragua from 2010 to 2012. Specimens were identified based on DNA sequence comparisons and an attempt was made to confirm whether L. acicola occurs in Central America.

MATERIALS AND METHODS

Collections used in the study

Specimens prepared from ex-type cultures and other representatives of all known Lecanosticta species and closely related species (Quaedvlieg et al. 2012) were obtained from the culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (CBS), and from the UK National Fungus Collection maintained by CABI Bioscience (Egham, UK: IMI). Living cultures or DNA of six isolates from Central America examined by Evans (1984), and believed to represent L. acicola, were also acquired from IMI (Table 1). Furthermore, isolates of Dothistroma septosporum, D. pini, Phaeophleospora eugenia, P. gregaria, and Amycosphaerella africana that represent genera in Mycosphaerellaceae closely related to Lecanosticta (Quaedvlieg et al. 2012) were included for comparative purposes. These cultures were obtained from CBS and the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) in Pretoria, South Africa (Table 1).

Table 1

Details of isolates used in this study

Species	CMW numbera	Other collection numberb	Sampling site (Country, Region, Location)	Host	Collection date	Collector	GenBank accession numbersd
							ITS	TEF1	BT1	MS204	RPB2
Amycosphaerella africana	45395	CBS 110843	South Africa, Western Cape Province, Pampoenvlei	Eucalyptus cladocalyx	Nov 1994	Crous PW	KF901702	JX901653	MK015047	MK015515	MK015290
A. africana	45396	CBS 680.95	South Africa, Western Cape Province, Stellenbosch mountain	E. viminalis	Oct 1994	Crous PW	AY626981	KF903117	MK015048	MK015516	MK015291
Dothistroma pini	10930	CBS 116485	USA, Michigan, Montcalm County, Crystal Lake	Pinus nigra	2001	Adams G, Barnes I	AY808301	AY808266	AY808196	NA	MK015292
D. pini	10951	CBS 116487	USA, Michigan, Montcalm County, Stanton	P. nigra	2001	Adams G, Barnes I	AY808302	AY808267	AY808197	NA	MK015293
D. septosporum	44656	CBS 140339	Russia, St. Petersburg, Park Sosnovka	P. sylvestris	Nov 2013	Drenkhan R, Musolin D, Adamson K	KU948400	MK015397	MK015049	MK015517	MK015294
D. septosporum	44657	CBS 141531	Russia, St. Petersburg, Park Sosnovka	P. sylvestris	Nov 2013	Drenkhan R, Musolin D, Adamson K	KU948401	MK015398	MK015050	MK015518	MK015295
Lecanosticta acicola	9985	CBS 871.95	France	P. radiata	Apr 1995	Morelet M	GU214663	MK015399	MK015051	MK015519	MK015296
L. acicola	45426	CBS133790	Lithuania	P. mugo	2009	Markovskaja S, Kacergius A, Treigiene A	HM367708	JX901645	MK015052	MK015520	MK015297
L. acicola	45427	CBS 133791	USA, New Hampshire, Blackwater	P. strobus	Jun 2011	Ostrofsky B	KC012999	KC013002	MK015053	MK015521	MK015298
L. acicola	45428	CBS 322.33	USA	P. palustris	Feb 1933	Siggers PV	MK015156	MK015400	MK015054	MK015522	MK015299
L. acicola	50541		Lithuania, Curonian Spit, Juodkrante	P. mugo	Sep 2014	Markovskaja S	MK015157	MK015401	MK015055	MK015523	MK015300
L. acicola	50542		Lithuania, Curonian Spit, Juodkrante	P. mugo	Sep 2014	Markovskaja S	MK015158	MK015402	MK015056	MK015524	MK015301
L. brevispora	- e	1A.N5S2	Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015159	MK015403	–	–	–
L. brevispora	- e	1C.N1S3	Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015160	MK015404	MK015057	NA	NA
L. brevispora	- e	1C.N5S4	Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015161	MK015405	MK015058	MK015525	MK015302
L. brevispora	- e	1C.N6S2	Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015162	MK015406	–	–	–
L. brevispora	- e	1D.N1S3	Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015163	MK015407	MK015059	NA	NA
L. brevispora	- e	IB31.4a	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015164	MK015408	MK015060	MK015526	MK015303
L. brevispora	36894		Guatemala, Finca La Soledad (near Jalapa), Mataquescuintla	P. pseudostrobus	Oct 2010	Barnes I	MK015165	MK015409	MK015061	NA	MK015304
L. brevispora	37123		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015166	MK015410	NA	NA	MK015305
L. brevispora	42646		Honduras	P. oocarpa	–	–	MK015167	MK015411	MK015062	MK015527	MK015306
L. brevispora	42647		Guatemala, Lugar, La Soledad, Jalapa	P. oocarpa	Oct 2010	Barnes I	MK015168	MK015412	MK015063	MK015528	MK015307
L. brevispora	45424	CBS 133601	Mexico	Pinus sp.	Oct 2009	Yanes-Morales M	JX901763	JX901649	MK015064	MK015529	MK015308
L. brevispora	46499		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015169	MK015413	–	–	–
L. brevispora	46500		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015170	MK015414	–	–	–
L. brevispora	46501		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015171	MK015415	MK015065	NA	NA
L. brevispora	46502		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015172	MK015416	–	–	–
L. brevispora	46503		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015173	MK015417	MK015066	MK015530	MK015309
L. brevispora	46504		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015174	MK015418	MK015067	MK015531	MK015310
L. brevispora	46505		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015175	MK015419	NA	NA	MK015311
L. brevispora	46506		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015176	MK015420	–	–	–
L. brevispora	46507		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015177	MK015421	–	–	–
L. brevispora	46508		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015178	MK015422	–	–	–
L. brevispora	46509		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015179	MK015423	–	–	–
L. brevispora	46510		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015180	MK015424	NA	NA	MK015312
L. brevispora	46511		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015181	MK015425	–	–	–
L. brevispora	46512		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015182	MK015426	–	–	–
L. brevispora	46807		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015183	MK015427	MK015068	MK015532	MK015313
L. brevispora	49291		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015184	MK015428	MK015069	NA	MK015314
L. brevispora	49292		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015185	MK015429	MK015070	MK015533	MK015315
L. brevispora	49293		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015186	NA	MK015071	MK015534	MK015316
L. brevispora	49294		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015187	NA	MK015072	MK015535	MK015317
L. brevispora	49295		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015188	NA	MK015073	MK015536	MK015318
L. brevispora	49296		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015189	MK015430	MK015074	MK015537	MK015319
L. brevispora	49297		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015190	MK015431	MK015075	MK015538	MK015320
L. brevispora	49298		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015191	MK015432	MK015076	MK015539	MK015321
L. brevispora	50523		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015192	MK015433	–	–	–
L. brevispora	50526		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015193	MK015434	MK015077	NA	NA
L. brevispora	50527		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015194	MK015435	NA	NA	MK015322
L. brevispora	50528		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015195	MK015436	MK015078	NA	NA
L. brevispora	50529		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015196	MK015437	–	–	–
L. brevispora	50530		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015197	MK015438	MK015079	MK015540	MK015323
L. brevispora	50531		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015198	MK015439	MK015080	MK015541	MK015324
L. brevispora	50532		Guatemala, Chimaltenango, Tecpán, Finca La Esperanza	P. pseudostrobus	Jun 2011	Barnes I	MK015199	MK015440	–	–	–
L. brevispora	51050		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015200	MK015441	NA	MK015542	MK015325
L. gloeospora c	42645	IMI 283812	Mexico, Nuevo León, Iturbide-Galeana	P. pseudostrobus	May 1983	Evans HC	KU948431	MK015442	MK015081	MK015543	MK015326
L. guatemalensis	- e	IB30/2d	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015201	MK015443	–	–	–
L. guatemalensis	- e	IB32/1a	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015202	MK015444	–	–	–
L. guatemalensis	- e	IB32/2e	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015203	MK015445	MK015082	NA	NA
L. guatemalensis	- e	IB35/2e	Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015204	MK015446	MK015083	NA	NA
L. guatemalensis	- e	IB35/2j	Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015205	MK015447	–	–	–
L. guatemalensis	- e	IB35/9a	Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015206	MK015448	MK015084	NA	NA
L. guatemalensis c		IMI 275573	Honduras, Yoro	P. oocarpa	Oct 1980	Evans HC	MK015207	MK015449	NA	NA	NA
L. guatemalensis c		IMI 281563	Honduras	P. caribaea	May 1982	Evans HC	MK015208	NA	NA	NA	NA
L. guatemalensis c		IMI 281596	Nicaragua	P. tecunumanii	Nov 1981	Evans HC	MK015209	MK015450	NA	NA	NA
L. guatemalensis	- e	N3/1c	Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015210	MK015451	MK015085	MK015544	MK015327
L. guatemalensis	36811		Guatemala, Jalapa, Finca Forestal Soledad	P. maximinoi	Oct 2010	Barnes I	MK015211	MK015452	MK015086	NA	MK015328
L. guatemalensis	36812		Guatemala, Coban, San Juan Chamelco	P. maximinoi	Oct 2010	Barnes I	MK015212	MK015453	MK015087	MK015545	MK015329
L. guatemalensis	37121		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015213	MK015454	–	–	–
L. guatemalensis	37122		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015214	MK015455	MK015088	MK015546	MK015330
L. guatemalensis	37124		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015215	MK015456	–	–	–
L. guatemalensis	37126		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015216	MK015457	MK015089	MK015547	MK015331
L. guatemalensis	37127		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015217	MK015458	–	–	–
L. guatemalensis c	42206	IMI 281598	Guatemala	P. oocarpa	1983	Evans HC	JX901764	JX901650	MK015090	MK015548	MK015332
L. guatemalensis	43890		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015218	MK015459	–	–	–
L. guatemalensis	43891		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015219	MK015460	MK015091	NA	NA
L. guatemalensis	43892		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015220	MK015461	MK015092	NA	NA
L. guatemalensis	43893		Guatemala, Chiquimula, San José la Arada	P. oocarpa	Oct 2010	Barnes I	MK015221	MK015462	MK015093	NA	NA
L. guatemalensis	43894		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015222	MK015463	MK015094	NA	NA
L. guatemalensis	43895		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015223	MK015464	MK015095	MK015549	MK015333
L. guatemalensis	45386		Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015224	MK015465	–	–	–
L. guatemalensis	45387		Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015225	MK015466	MK015096	MK015550	MK015334
L. guatemalensis	45391		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015226	MK015467	MK015097	NA	NA
L. guatemalensis	45392		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2011	Barnes I	MK015227	MK015468	MK015098	MK015551	MK015335
L. guatemalensis	45393		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015228	MK015469	–	–	–
L. guatemalensis	45394		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015229	NA	–	–	–
L. guatemalensis	46811		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015230	MK015470	MK015099	MK015552	MK015336
L. guatemalensis	46817		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015231	MK015471	MK015100	MK015553	MK015337
L. guatemalensis	46819		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015232	NA	–	–	–
L. guatemalensis	47108		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015233	MK015472	–	–	–
L. guatemalensis	49400		Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015234	MK015473	MK015101	MK015554	MK015338
L. guatemalensis	49402		Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015235	MK015474	MK015102	MK015555	MK015339
L. guatemalensis	51052		Guatemala, Chiquimula, San José la Arada	P. oocarpa	Oct 2010	Barnes I	MK015236	MK015475	MK015103	MK015556	MK015340
L. guatemalensis	51142		Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015237	MK015476	MK015104	MK015557	MK015341
L. jani	- e	267.44.N1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015238	MK015477	MK015105	MK015558	MK015342
L. jani	- e	267.47.N1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015239	MK015478	MK015106	MK015559	MK015343
L. jani	- e	267.47.N2	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015240	MK015479	MK015107	MK015560	MK015344
L. jani	- e	267.51.N2S1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015241	MK015480	NA	NA	MK015345
L. jani	- e	267.52.N1S1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015242	MK015481	MK015108	MK015561	MK015346
L. jani	- e	267.52.N2S1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015243	MK015482	MK015109	MK015562	MK015347
L. jani	- e	IB30/2b	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015244	MK015483	MK015110	MK015563	NA
L. jani	- e	IB35/3c	Guatemala, Chiquimula	P. oocarpa	Oct 2010	Barnes I	MK015245	MK015484	MK015111	MK015564	MK015348
L. jani	- e	IB13/2f	Guatemala	P. maximinoi	Oct 2010	Barnes I	MK015246	MK015485	MK015112	MK015565	MK015349
L. jani	- e	N3/2c	Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015247	NA	MK015113	MK015566	MK015350
L. jani	36808		Guatemala, Jalapa, Finca Forestal Soledad	P. maximinoi	Oct 2010	Barnes I	MK015248	NA	MK015114	MK015567	MK015351
L. jani	36810		Guatemala, Jalapa, Finca Forestal Soledad	P. maximinoi	Oct 2010	Barnes I	MK015249	NA	MK015115	MK015568	MK015352
L. jani	37128		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015250	MK015486	MK015116	MK015569	MK015353
L. jani	38950	CBS 144446; PREM 62186	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015251	MK015487	MK015117	MK015570	MK015354
L. jani	38958	CBS 144456; PREM 62185	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015252	MK015488	MK015118	MK015571	MK015355
L. jani	38959		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015253	NA	NA	NA	NA
L. jani	38968		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015254	NA	MK015119	NA	NA
L. jani	45388		Guatemala	P. maximinoi	Oct 2010	Barnes I	MK015255	NA	MK015120	MK015573	MK015356
L. jani	45389		Guatemala	P. maximinoi	Oct 2010	Barnes I	MK015256	MK015489	MK015121	MK015574	MK015357
L. jani	47109		Guatemala	P. maximinoi	Oct 2010	Barnes I	MK015257	MK015490	MK015122	MK015575	MK015358
L. jani	48830		Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015258	NA	MK015123	MK015576	MK015359
L. jani	48831	CBS 144447; PREM 62187	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015259	MK015491	MK015124	MK015577	MK015360
L. jani	49401		Guatemala	P. maximinoi	Oct 2010	Barnes I	MK015260	MK015492	NA	MK015578	MK015361
L. jani	51051		Guatemala	P. maximinoi	Oct 2010	Barnes I	MK015261	MK015493	MK015125	MK015579	MK015362
L. jani	51058		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015262	MK015494	MK015126	MK015580	MK015363
L. jani	51059		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. tecunumanii	Sep 2012	Barnes I	MK015263	MK015495	MK015127	MK015581	MK015364
L. jani	51143		Nicaragua, Matagalpa	P. oocarpa	Jun 2011	Barnes I	MK015264	NA	MK015128	MK015582	MK015365
L. longispora	45429	CBS 133602	Mexico	Pinus sp.	Oct 2009	Yanes-Morales M	JX901766	JX901651	MK015129	MK015583	MK015366
L. longispora	45430	CPC 17941	Mexico	Pinus sp.	Oct 2009	Yanes-Morales M	JX901765	JX901652	MK015130	MK015584	MK015367
L. pharomachri	- e	267.8A.N2S1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015265	MK015496	NA	NA	MK015368
L. pharomachri	- e	267.12.N1S2	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015266	NA	NA	NA	MK015369
L. pharomachri	- e	267.30.MD.N1	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015267	NA	NA	NA	MK015370
L. pharomachri	- e	267.30.MD.N2	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015268	MK015497	MK015131	NA	MK015371
L. pharomachri	- e	267.30.N4	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015269	MK015498	MK015132	MK015585	MK015372
L. pharomachri	37132		Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015270	MK015499	MK015133	MK015586	MK015373
L. pharomachri	37133		Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015271	MK015500	MK015134	MK015587	MK015374
L. pharomachri	37134		Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015272	MK015501	MK015135	MK015588	MK015375
L. pharomachri	37136	CBS 144448; PREM 62188	Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015273	MK015502	MK015136	MK015589	MK015376
L. pharomachri	38947	CBS 144695; PREM 62189	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015274	MK015503	MK015137	MK015590	MK015377
L. pharomachri	38974	CBS 144449; PREM 62190	Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015275	MK015504	MK015138	MK015591	MK015378
L. pharomachri	38975		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015276	NA	NA	NA	NA
L. pharomachri	38976		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015277	MK015505	MK015139	NA	MK015379
L. pharomachri	46810		Honduras	P. oocarpa	–	–	MK015278	MK015506	MK015140	MK015592	MK015380
L. pharomachri	46813		Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015279	MK015507	MK015141	MK015593	MK015381
L. pharomachri	51053		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015280	NA	MK015142	NA	MK015382
L. pharomachri	51054		Guatemala, Jalapa, Finca La Soledad, Mataquescuintla	P. oocarpa	Sep 2012	Barnes I	MK015281	MK015508	NA	NA	MK015383
L. tecunumanii	46805	CBS 144450; PREM 62191	Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015282	MK015509	MK015143	MK015594	MK015384
L. tecunumanii	46812	CBS 144452; PREM 62193	Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015283	MK015510	MK015144	MK015595	MK015385
L. tecunumanii	49403	CBS 144451; PREM 62192	Guatemala, Baja Verapaz, San Jerónimo, Salamá	P. tecunumanii	Oct 2010	Barnes I	MK015284	MK015511	MK015145	MK015596	MK015386
L. variabilis	36809	CBS 144455; PREM 62195	Guatemala, Jalapa, Finca Forestal Soledad	P. maximinoi	Oct 2010	Barnes I	MK015285	MK015512	MK015146	MK015597	MK015387
L. variabilis	37125	CBS 144454; PREM 62194	Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015286	KJ938446	MK015147	MK015598	MK015388
L. variabilis	37129		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015287	KJ938445	MK015148	MK015599	MK015389
L. variabilis c	42205	IMI 281561; CBS 144453; PREM 62196	Honduras, Santa Barbara, Lago Yojoa	P. caribaea	Oct 1980	Evans HC	MK015288	MK015513	MK015149	MK015600	MK015390
L. variabilis	45390		Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc	P. oocarpa	Oct 2010	Barnes I	MK015289	MK015514	MK015150	MK015601	MK015391
L. variabilis	45425	CBS 133789	Mexico	Pinus sp.	Nov 2009	Yanez-Morales M	JX901762	JX901648	MK015151	MK015602	MK015392
Phaeophleospora eugeniae	45432	CPC15159	Brazil, Vicosa, Paraiso	Eugenia uniflora	Mar 2008	Alfenas AC	FJ493189	JX901667	MK015152	MK015603	NA
P. eugeniae	45433	CPC 15143	Brazil, Vicosa, Paraiso	E. uniflora	Mar 2008	Alfenas AC	FJ493188	JX901666	MK015153	MK015604	NA
P. gregaria	45434	CBS 111166	South Africa, Western Cape Province, de Hoop Nature Reserve	Eucalyptus cladocalyx	Sep 1995	Wood A	JX901773	JX901664	MK015154	MK015605	MK015393
P. gregaria	45435	CBS 114662	South Africa, Western Cape Province, Devon Valley, Stellenbosch	Eucalyptus sp.	Jun 1995	Crous PW	DQ302953	JX901654	MK015155	MK015606	MK015394

aCMW Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa;

bCBS Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, CPC Personal collection of Pedro Crous housed at CBS, IMI The UK National Fungus Collection, CABI Bioscience, Egham, UK, PREM The dried herbarium collection of the South African National Collection of Fungi, Mycology Unit, Biosystematics Division, Plant Protection Institute, Agricultural Research Council, Pretoria, South Africa

cCultures were collected by HC Evans in Central America

d‘-‘= was not amplified; ‘NA‘= amplification unsuccessful;

e = no viable culture available that could be submitted to CMW

Ex-type isolate of each species is indicated in bold

Pine needles, showing symptoms of brown spots or bands, were collected from Pinus species native to Central America from 2010 to 2012 in Guatemala, as well as from Honduras and Nicaragua in 2011 (Table 1). Conidiomata formed on the needles were aseptically excised, rolled onto 2% Dothistroma Sporulating Media (DSM: 5 g yeast extract (Biolab, Merck, Modderfontein, South Africa), 20 g malt extract (Biolab) and 15 g agar (BD Difco™, Sparks, MD) per litre of distilled water) with 100 mg/L streptomycin (Sigma-Aldrich, St Louis, MO) in order to release conidia from the conidiomata as described by Barnes et al. (2004). The isolated conidiomata were incubated for one to two days at 23 °C. The plates were examined using a dissection microscope and single germinating conidia were selected and replated onto 2% DSM. The single conidial isolates were grown for 4–6 wk. on a natural day light cycle, at 23 °C.

DNA extractions and sequencing

Fungal tissue was scraped from the surface of the cultures on 2% DSM with a sterile scalpel blade and lyophilized. The freeze-dried mycelium was homogenized using a Retsch MM301 mixer mill (Haan, Germany) and approximately 20 ng of the crushed mycelium was used as starting material for DNA extractions. DNA was extracted using a Zymo Research ZR Fungal/Bacterial DNA MiniPrep™ kit (Irvine, CA) and eluted into a final volume of 50 μl. The quality and quantity of the extracted DNA was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA). DNA concentrations were diluted to 20 ng/μl working stock for polymerase chain reaction (PCR) amplifications and stored at − 20 °C until further use.

The nuclear rDNA region encompassing the internal transcribed spacers (ITS) 1 and 2, along with the 5.8S rDNA region was amplified using primers ITS1 and ITS4 (White et al. 1990) and a portion of the translation elongation factor 1-α gene (TEF1) using primers EF1-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998) for all the isolates. The Beta-tubulin-2 gene region (BT2) was amplified using the primer pair T1 (O’Donnell and Cigelnik 1997) and β-Sandy-R (Stukenbrock et al. 2012) or the primers Bt2A and Bt2B (Glass and Donaldson 1995). The Beta-tubulin-1 gene region (BT1) was amplified using primers Bt1A and Bt1B (Glass and Donaldson 1995), the RNA polymerase II second largest subunit (RPB2) gene region using primers RPB2-5f2 (Sung et al. 2007) and RPB2-7cR (Liu et al. 1999) and the guanine nucleotide-binding protein subunit beta (MS204) using primers MS204F.cerato and MS204R.cerato (Fourie et al. 2015).

PCR reactions for each of the six regions contained 20 ng DNA, 2.5 μl 10x PCR reaction buffer, 2.5 mM MgCl2, 400 nM of each primer, 200 μM of each dNTP and 1 U Faststart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN). Reaction volumes were adjusted to 25 μl with sterile SABAX water (Adcock Ingram, Midrand, South Africa). PCR reactions were carried out on an Applied Biosystems® Veriti® 96 well Thermal cycler (Thermo Fisher Scientific, Waltham, MA). The cycling conditions for all six gene regions included an initial denaturation step at 95 °C for 4 min, 10 cycles consisting of 94 °C for 20 s (denaturation), a 45 s annealing step according to the primer pair annealing temperature (Table 2) and an elongation step of 45 s at 72 °C. This was followed by a further 25 cycles of 94 °C for 20 s, 45 s with a 5 s extension step per cycle at the indicated annealing temperature, a 72 °C extension for 45 s and a final step of 72 °C for 10 min. The annealing temperature was set at 56 °C for ITS, 52 °C for TEF1, 50 °C for BT1, 52 °C for BT2, 55 °C for MS204 and 56 °C for RPB2. To visualise amplified products, 5 μl PCR products were stained with 1 μl GelRed™ nucleic acid gel stain (Biotium, Fremont, CA) and separated on 2% SeaKem® LE agarose gel (Lonza, Rockland, ME) for 20 min at 100 V and viewed under a UV light using the GelDoc™ EZ Imager (BioRad, Hercules, CA). PCR products were cleaned with a 6.65% G-50 Sephadex solution (Sigma-Aldrich, St Louis, MO) following the manufacturer’s instructions using Centri-sep spin columns (Princeton Separations, Freehold, NJ).

Table 2

Primers used for PCR amplification and sequencing in this study

Locus	Primer name	Direction	Primer sequence 5′ to 3’	Annealing temperature used (°C)	Amplification success	Reference
BT1	Bt1a	Forward	TTC CCC CGT CTC CAC TTC TTC ATG	50	87.4%	Glass and Donaldson 1995
	Bt1b	Reverse	GAC GAG ATC GTT CAT GTT GAA CTC	50		Glass and Donaldson 1995
BT2a	T1	Forward	AAC ATG CGT GAG ATT GTA AGT	52	–	O’Donnell and Cigelnik 1997
	β-Sandy-R	Reverse	GCR CGN GGV ACR TAC TTG TT	52		Stukenbrock et al. 2012
	Bt2a	Forward	GGT AAC CAA ATC GGT GCT GCT TTC	52	–	Glass and Donaldson 1995
	Bt2b	Reverse	ACC CTC AGT GTA GTG ACC CTT GGC	52		Glass and Donaldson 1995
TEF1	EF1-728F	Forward	CAT CGA GAA GTT CGA GAA GG	52	88.2%	Carbone and Kohn 1999
	EF-2	Reverse	GGA RGT ACC AGT SAT CAT GTT	52		O’Donnell et al. 1998
ITS	ITS1	Forward	GAA GTA AAA GTC GTA ACA AGG	56	100%	White et al. 1990
	ITS4	Reverse	TCC TCC GCT TAT TGA TAT GC	56		White et al. 1990
MS204	MS204F.cerato	Forward	AAG GGC ACC CTC GAG GGC CAC	55	71.7%	Fourie et al. 2015
	MS204R.cerato	Reverse	GAT GGT RAC GGT GTT GAT GTA	55		Fourie et al. 2015
RPB2	RPB2-5f2	Forward	GGG GWG AYC AGA AGA AGG C	56	82.7%	Sung et al. 2007
	fRPB2-7cR	Reverse	CCC ATR GCT TGY TTR CCC AT	56		Liu et al. 1999

aBT2 amplification success using all primer combinations was very low and abandoned

The concentrations of the cleaned PCR products were determined using a NanoDrop ND-1000 spectrophotometer and 60–100 ng of DNA and products were sequenced in both the forward and reverse direction using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on an ABI PRISM 3500xl capillary auto sequencer (Thermo Fisher Scientific).

Forward and reverse sequences were aligned and consensus sequences generated in CLC Main workbench version 8.0 (CLC Bio, https://www.qiagenbioinformatics.com/products/clc-main-workbench/). All consensus sequences generated in this study were deposited in GenBank that is hosted by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/genbank/) (Table 1).

Data analyses

Five datasets (BT1, ITS, MS204, RPB2 and TEF1) were generated and analysed individually. A partition homogeneity test (PHT) was performed with the software package PAUP* 4.0b10 (Swofford 2003) to test congruence between the five gene regions and a sixth dataset, where sequences were available for all five gene regions, was compiled and analysed. The BT1, ITS, MS204 and RPB2 datasets included all of the sequences generated in this study and additional sequences available from GenBank (Table 1). The TEF1 dataset included all of the sequence data generated in this study as well as additional sequences representing 14 different TEF1 haplotypes of L. acicola (including possible cryptic species) (Janoušek et al. 2016) that were downloaded from GenBank (Table 3). Sequences for all datasets were aligned with the online version of MAFFT Version 7 (Katoh and Standley 2013; http://mafft.cbrc.jp/alignment/server/) using default settings. Aligned data were imported into MEGA 7.0.14 (Kumar et al. 2016) and manually checked and adjusted.

Table 3

GenBank numbers of Lecanosticta acicola TEF1 haplotypes included in the TEF1 phylogenetic analysis (Fig. 2) as well as additional locations represented by the haplotypes

Species name assigned in this studya	GenBank Accession number	Country	State / Region	Location	Host	Date of collection	Collector / Supplier
Lecanosticta acicola	KJ938442	Japan	Shimane	Matsue, Hamanogi	Pinus thunbergii	Feb 2010	Suto Y
L. acicola	KJ938439	Mexico	Nuevo León	Iturbide, Bosque Escuela	Pinus halepensis	May 2010	Marmolejo JG
L. acicola	KJ938440	Mexico	Nuevo León	Iturbide, Bosque Escuela	Pinus halepensis	May 2010	Marmolejo JG
L. acicola	KJ938441	Mexico	Nuevo León	Iturbide, Bosque Escuela	Pinus halepensis	May 2010	Marmolejo JG
L. acicola	KJ938438	USA	Maine	York, Lyman	Pinus strobus	Jun 2011	Ostrofsky W
L. acicola	KJ938443	USA	Mississippi	Harrison County	Pinus palustris	Oct 2012	Bartlett B, Burdine C
L. acicola	KJ938444	USA	Mississippi	Harrison County	Pinus palustris	Oct 2012	Bartlett B, Burdine C
L. acicola	KJ938450	USA	Mississippi	Harrison County	Pinus palustris	Oct 2012	Bartlett B, Burdine C, Roberds J
L. acicola	KJ938451	USA	Mississippi	Harrison County	Pinus palustris	Oct 2012	Bartlett B, Burdine C
Lecanosticta variabilis	KJ938445	Guatemala	Alta Verapaz	Santa Cruz Verapaz, near Tactíc	Pinus oocarpa	Oct 2010	Barnes I
L. variabilis	KJ938446	Guatemala	Alta Verapaz	Santa Cruz Verapaz, near Tactíc	Pinus oocarpa	Oct 2012	Barnes I
L. variabilis	KJ938447	Mexico	Nuevo León	Piñal de los Amoles, Querétaro	Pinus sp.	2011	Kunte L
L. variabilis	KJ938448	Mexico	Nuevo León	Iturbide, Bosque Escuela	Pinus halepensis	May 2010	Marmolejo JG
L. variabilis	KJ938449	Mexico	Nuevo León	Galeana, Cerro del Potosí	Pinus arizonica var. stormiae	Apr 2010	Marmolejo JG
Countries, regions, locations and hosts represented by the above isolatesb
	the same as KJ938438	Austria	Lower Austria	Hollenstein an der Ybbs	Pinus mugo	Oct 2004	Kirisits T, Barnes I
	the same as KJ938438	Austria	Lower Austria	Opponitz	Pinus mugo	2010	Hintsteiner M
	the same as KJ938438	Austria	Lower Austria	Saimannslehen	Pinus sp.	2010	Hintsteiner M
	the same as KJ938438	Austria	Lower Austria	Sankt Gallen	Pinus mugo	2010	Hintsteiner M
	the same as KJ938438	Austria	Lower Austria	Steyer, Pestalozzistraße	Pinus mugo	2010	Hintsteiner M
	the same as KJ938438	Austria	Lower Austria	Waidehofen an der Ybbs	Pinus mugo	Aug 2010	Janoušek J
	the same as KJ938438	Austria	Upper Austria	Gmunden	Pinus nigra	Jun 2012	Kirisits T
	the same as KJ938438	Canada	Québec	Demers-Centre	Pinus strobus	Jun 2011	Harvey L
	the same as KJ938438	Canada	Québec	Lake Aberdeen	Pinus strobus	Jun 2011	Harvey L
	the same as KJ938438	Canada	Québec	Lake Pinseault	Pinus strobus	Jun 2011	Harvey L
	the same as KJ938438	Canada	Québec	Montréal	Pinus mugo	Jun 2011	Harvey R
	the same as KJ938438	Canada	Québec	Waltham	Pinus strobus	Jun 2011	Harvey L
	the same as KJ938442	China	Fujie		Pinus elliottii	1988	Zheng-Yu H
	the same as KJ938451	Colombia	Refocosta L-75	Villanueva, Casanare	Pinus caribaea	Mar 2011	Rodas C, Barnes I
	the same as KJ938438	Croatia		Zadar	Pinus halapensis	Sep 2009	Diminic D
	the same as KJ938438	Czech Republic	Southern Bohemia	Borkovická Blata	Pinus uncinata subsp. uliginosa	Oct 2011	Janoušek J
	the same as KJ938438	Czech Republic	Southern Bohemia	Červená Blata	Pinus uncinata subsp. uliginosa	Aug 2009	Dvořák M, Janoušek J
	the same as KJ938438	Estonia	Harju maakond	Tallin	Pinus ponderosa	Jul 2008	Cech T
	the same as KJ938451	France	Pyrénées-Atlantiques		Pinus radiata	2012	Kersaudy E, Ioos R
	the same as KJ938438	Germany	Bavaria	Grassau	Pinus mugo	2000	Blaschke FR, Wulf
	the same as KJ938438	Germany	Bavaria	Murnau	Pinus mugo	Feb 2010	Nannig A
	the same as KJ938438	Germany	Bavaria	Murnauer Filze	Pinus mugo	Nov 2011	Nannig A
	the same as KJ938438	Germany	Bavaria	Pfrűhlmoos	Pinus mugo	Nov 2011	Nannig A
	the same as KJ938438	Italy	Brecia	Gardone	Pinus mugo	Jun 2008	Cech T
	the same as KJ938438	Lithuania	Klaipėdský kraj	Curonian Spit, Juodkrante	Pinus mugo	2010	Markovskaja S
	the same as KJ938438	Slovenia	Upper Carniola	Bled	Pinus mugo	Jul 2009	Jurc D
	the same as KJ938442	South Korea	Naju	Sanpo-myeon	Pinus thunbergii	2010	KACC, Seo ST
	the same as KJ938451	Spain	Cantabria	San Sebastián de Garabandal	Pinus radiata	Oct 2012	Jankovský L, Janoušek J
	the same as KJ938438	Switzerland	Canton St Gallen	Walensee	Pinus mugo	Oct 1999	Wulf
	the same as KJ938438	USA	Maine	Androscoggin, Leeds	Pinus strobus	Jun 2011	Ostrofsky W
	the same as KJ938438	USA	Maine	Piscataquis, Sangerville	Pinus strobus	Jun 2011	Weimer J
	the same as KJ938438	USA	Maine	York, Lyman	Pinus strobus	Jun 2011	Ostrofsky W
	the same as KJ938438	USA	Michigan	Wexford County, Springville Township	Pinus sylvestris	2011	Odonnell J
	the same as KJ938444	USA	Mississippi	Harrison County	Pinus palustris	Oct 2012	Bartlett B, Burdine C, Roberds J
	the same as KJ938438	USA	New Hampshire	Hillsboro, Fox State Park	Pinus strobus	Jun 2011	Weimer J
	the same as KJ938438	USA	New Hampshire	Merrimack, Black Water Reserve	Pinus strobus	Jun 2011	Weimer J
	the same as KJ938438	USA	New Hampshire	Merrimack, Hopkinton-Everett	Pinus strobus	Jun 2011	Weimer J
	the same as KJ938438	USA	Vermont	Washington, Waterbury	Pinus strobus	Jun 2011	Lackey J
	the same as KJ938438	USA	Vermont	Windsor, Bethel	Pinus strobus	Jul 2011	Munck I
	the same as KJ938438	USA	Wisconsin	Merrillan	Pinus sylvestris	Apr 2010	Stanosz G

aLecanosticta variabilis was previously identified as L. acicola but is now defined as a new species

bInformation adapted from Janoušek et al. (2016), Table S1

Fig. 2

Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the TEF1 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “∆” next to the isolate indicates that the isolate either exhibits Type 2 morphology and groups with Subclade 1, or, exhibits Type 1 morphology and groups with Subclade 2

Three separate analyses were performed for each of the six datasets: Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). The MP analysis were performed with the software package PAUP* 4.0b10 (Swofford 2003). Gaps were treated as a fifth character state. One thousand random stepwise addition heuristic searches were performed with tree-bisection-reconnection (TBR) as the branch-swapping algorithm. Uninformative characters were excluded and the consistency index (CI), homoplasy index (HI), rescaled consistency index (RC), retention index (RI) and tree length (TL) were determined for the resulting trees (Table 4). The confidence levels were estimated by performing 1000 bootstrap replicates.

Table 4

PCR amplification size, phylogenetic data and the substitution models used in the phylogenetic analysis for each gene region and for the combined datasets

	ITS	TEF1	BT1	MS204	RPB2	Combined datasets
Approximate amplicon size (bp)	550	520	420	760	940	–
Number of taxa analysed	153	147	111	91	105	76
Aligned characters (bp)	734	586	440	785	929	3344
Number of parsimony-uninformative characters	621	143	357	519	538	2438
Number of parsimony-informative characters	114	423	82	266	371	1121
Number of trees retained	108	396	1	2448	420	100
Consistency index	0.865	0.499	0.739	0.791	0.738	0.607
Homoplasy index	0.135	0.501	0.261	0.209	0.262	0.393
Rescaled consistency index	0.850	0.459	0.703	0.748	0.696	0.555
Retention index	0.982	0.919	0.951	0.946	0.943	0.914
Tree Length	163	1675	138	546	722	2642
Substitution model	TPM2uf + G	GTR + G	GTR + G	TVM + G	TrN + G	GTR + G

In order to determine the ML and BI, the best fit substitution model for each of the data sets were determined using jModelTest 0.1.1 (Posada 2008). Maximum likelihood analysis was performed with the program PhyML 3.0 (Guindon et al. 2010). The confidence levels were estimated with 1000 bootstrap replicates.

MrBayes 3.1.2 (Ronquist et al. 2012) was used to determine the BI for each data set by applying the Markov Chain Monte Carlo (MCMC) method. For each dataset, four independent MCMC chains were randomly started and run for six million generations, applying the best substitution model determined by jModelTest 0.1.1. Trees were sampled every 100 generations. Burn-in values were determined using Tracer 1.6 (Rambaut et al. 2014) by comparing the log likelihoods. Trees sampled in the burn-in phase were discarded. The remaining trees were used to construct majority rule consensus trees and to determine posterior probabilities for the tree topology.

Morphological characterization

Cultures were grown on 2% Malt Extract Agar (MEA), Oatmeal Agar (OA) and Potato Dextrose Agar (PDA) (Crous et al. 2009b; Quaedvlieg et al. 2012) at 20 °C for 2 wk. in darkness in order to examine the morphology and colour of the cultures of each species. Cultures on MEA were used for microscopic measurements of the conidiophores, conidiogenous cells and conidia. Slides were mounted in SABAX water (Adcock Ingram, Midrand, South Africa) for microscopy and examined using a Zeiss Axioskop 2 Plus compound microscope (Zeiss, Oberkochen, Germany). Photographic images were captured with a Nikon DS-Ri2 camera with the NIS Element BR v4.3 software package (Nikon, Tokyo, Japan). Up to 50 measurements of each morphologically characteristic structure was taken for each ex-type isolate and ten measurements were made for each of the paratypes examined. The mean, standard deviation, minimum and maximum were calculated for each morphological structure and the measurements presented as (minimum–) (mean – standard deviation) – (mean + standard deviation) (−maximum) for the conidia and conidiogenous cells. For the conidiophores, the maximum observed length was indicated together with the width as (minimum–) (mean) (−maximum).

Temperature requirements for growth in culture was studied on representative isolates selected for each of the novel species. Four by four millimeter blocks of each culture were plated, in triplicate, onto the centres of 2% MEA plates per temperature (10, 15, 20, 25, and 30 °C) and incubated in darkness. The diameters of each colony were recorded weekly along perpendicular axes for 4 wk. The colour and shape of each colony was recorded after 2 wk. of growth at 20 °C. Culture colour was determined using Rayner’s colour chart (Rayner 1970).

Accession of cultures and types

Holotype specimens of the new species, which are dried cultures, are deposited in the National Mycological Herbarium in Pretoria (PREM). Cultures are deposited in the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, and ex-type cultures, as well as all other isolates included in this study, are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) in Pretoria, South Africa (Table 5).

Table 5

Specimens for which the morphology was examined for the description of Lecanosticta jani, L. pharomachri, L. tecunumanii and L. variabilis

Species	CMW numbera	Status of specimen	Herbarium specimenb	Ex-type isolatesc
Lecanosticta jani	CMW 38950d	Paratype	PREM 62186	CBS 144446
	CMW 38958d	Holotype	PREM 62185	CBS 144456
	CMW 48831e	Paratype	PREM 62187	CBS 144447
	CMW 51058d	Additional material examined
	CMW 51059d	Additional material examined
	CMW 51143e	Additional material examined
	CMW47109e	Additional material examined
Lecanosticta pharomachri	CMW 37136	Holotype	PREM 62188	CBS 144448
	CMW 38947	Paratype	PREM 62189	CBS 144695
	CMW 38974	Paratype	PREM 62190	CBS 144449
	CMW 38976	Additional material examined
	CMW 51053	Additional material examined
	CMW 51054	Additional material examined
Lecanosticta tecunumanii	CMW 46805	Holotype	PREM 62191	CBS 144450
	CMW 46812	Paratype	PREM 62193	CBS 144452
	CMW 49403	Paratype	PREM 62192	CBS 144451
Lecanosticta variabilis	CMW 42205	Holotype	PREM 62196	CBS 144453, IMI 281561
	CMW 37125	Paratype	PREM 62194	CBS 144454
	CMW 36809	Paratype	PREM 62195	CBS 144455
	CMW 45425	Additional material examined	CBS H-21112	CBS 133789
	CMW 37129	Additional material examined

aCMW Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; bThe herbarium deposits are dried cultures that serve as holotype and paratype specimens. PREM = The dried herbarium collection of the South African National Collection of Fungi, Mycology Unit, Biosystematics Division, Plant protection Institute, Agricultural Research Council, Pretoria, South Africa; cThe ex-type cultures are living cultures linked to the holotype and paratype specimens. CBS = The culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; IMI = The UK National Fungus Collection maintained by CABI Bioscience, Egham, UK; d Lecanosticta jani cultures with the Type 2 morphology; e Lecanosticta jani cultures with the Type 1 morphology

RESULTS

Fungal collections

Twenty-six isolates or DNA samples were obtained from culture collections to include in the study. An additional 127 isolates of putative Lecanosticta species were obtained from symptomatic needles collected from 36 different trees in Guatemala, Nicaragua and Honduras (Table 1). In Guatemala, 22 isolates were obtained from Pinus oocarpa, P. maximinoi, and P. tecunumanii needles that were collected in the Alta Verapaz District, 16 isolates were obtained from P. oocarpa needles collected in Chiquimula, 35 isolates from P. pseudostrobus needles collected in the Chimaltenango District in the Tecpán Municipality, eight isolates from P. tecunumanii needles collected in the Baja Verapaz District, 29 isolates from P. tecunumanii and P. oocarpa needles collected in the Jalapa District, and seven isolates from P. maximinoi needles in Coban and other regions (Table 1). Two isolates were obtained from P. oocarpa needles collected in Honduras and eight isolates were made from P. oocarpa needles collected in Matagalpa, Nicaragua.

DNA extraction and sequencing

The ITS and TEF1 regions were sequenced for all 153 isolates obtained and the BT1, MS204 and RPB2 regions were sequenced for 127 representatives of all monophyletic groups identified in the generated ITS and TEF1 phylogenetic trees. The selected representatives included all of the closely related Mycosphaerellaceae isolates, all the isolates that did not group with known Lecanosticta species, and a selection of isolates that grouped with known Lecanosticta species (Table 1). PCR fragments of approximately 550 bp were generated for ITS, 520 bp for TEF1, 420 bp for BT1, 760 bp for MS204 and 940 bp for RPB2. The amplification success of the TEF1, BT1, MS204 and RPB2 gene regions varied for the isolates that were selected and the amplification success rate of TEF1 was 88.2%, BT1 was 87.4%, MS204 was 71.7 and 82.7% for the RPB2 region (Table 2). The BT2 region did not amplify well across species of Lecanosticta. The amplification success rate and subsequent sequencing of the BT2 region using the T1 and β-Sandy-R primer pair, as well as Bt2a and Bt2b was very poor and further analysis of the BT2 region was abandoned.

Phylogenetic analyses

For the analyses, the datasets of the ITS region consisted of 153 taxa with 734 aligned nucleotides including gaps; the TEF1 dataset consisted of 147 taxa with 586 aligned nucleotides, the BT1 dataset consisted of 111 taxa with 440 aligned nucleotides; the MS204 dataset consisted of 91 taxa with 785 aligned nucleotides, and the RPB2 dataset consisted of 105 taxa with 929 aligned nucleotides, all including gaps. The PHT test yielded a P value = 0.01 and therefore the five datasets were considered incongruent. However, it was previously argued that a P value > 0.01 did not reduce phylogenetic accuracy (Cunningham 1997) and a combined phylogenetic tree representing the five gene regions ITS, TEF1, BT1, MS204 and RPB2 was constructed for presentation purposes (Fig. 1). The combined dataset consisted of 76 taxa with 3344 aligned nucleotides including gaps. Constant characters, parsimony-uninformative and informative characters, the consistency index (CI), homoplasy index (HI), rescaled consistency index (RC), retention index (RI) and tree length (TL) values for the maximum parsimony analyses are indicated in Table 4. For the parsimony analyses, 108 trees were retained for ITS, 396 for TEF1, 1 for BT1, 2448 for MS204 and 420 for RPB2. The best fit substitution models for ML and BI were selected by Akaike Information Criterion (AIC) and are indicated in Table 4. A 10% burn-in value was selected in the BI analysis for each of the data matrices for each of the analyses. Because the MP, ML and BI analysis all resulted in similar tree topologies, the ML trees were selected and chosen for presentation (Figs. 1 and 2, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4).

Fig. 1

Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the combined data of the ITS, TEF1, BT1, MS204 and RPB2 gene regions. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma septosporum was used as the outgroup taxa. The indicated clades are referred to in the text. All represented type species are indicated in bold and with a “T”

Phylogenetic analyses of the combined dataset (Fig. 1), ITS (Additional file 1: Figure S1), TEF1 (Fig. 2) and MS204 (Additional file 3: Figure S3) consistently grouped the isolates sequenced in this study into seven distinct clades. The clades in Fig. 2 and Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4 are labelled according to the clades assigned in Fig. 1. In the case of RPB2 (Additional file 4: Figure S4) Clades 1–4, and 7 were also present but Clades 5 and 6 were not distinct from each other for this particular gene region. In the case of BT1 (Additional file 2: Figure S2), Clades 3, 5 and 6 could not be distinguished from each other. None of the isolates grouped with the types of L. gloeospora or L. longispora.

Forty-two of the isolates from Central America grouped in Clade 1 based on the ITS analysis (Additional file 1: Figure S1) and were identified as Lecanosticta brevispora. This was the most common species identified from the Central American collection and most isolates were from Chimaltenango on Pinus pseudostrobus. The pathogen was also isolated from P. oocarpa needles near Jalapa as well as near Tactíc in Guatemala and in Honduras. This clade was well supported for all five of the gene regions analysed.

Twenty-seven isolates grouped into Clade 2 in the ITS analyses (Additional file 1: Figure S1) and represent an undescribed species. Clade 2 resolved into two subclades in the five gene analyses. Subclade 1 was mostly isolated from Chiquimula and Alta Verapaz in Guatemala on P. oocarpa, P. maximinoi and P. tecunumanii as well as from P. oocarpa in Nicaragua. Isolates collected in Jalapa in Guatemala mostly grouped into Subclade 2. However, the topology of isolates CMW 47109 (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4; Subclade 2 on Fig. 2), CMW 51059 (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4), IB30.2b (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3; Subclade 2 on Fig. 2) and IB30.2b (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4; Subclade 2 on Fig. 2) changed in the two subclades depending on the gene region analysed (Fig. 2, Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4). Furthermore, the two subclades were not well supported for the BT1 gene region. Therefore, the two subclades are treated here as representing a single species.

Clade 3 also represented an undescribed Lecanosticta species. This clade included 11 isolates from P. oocarpa in Jalapa, Guatemala, one isolate from P. oocarpa in Honduras, as well as five isolates collected from Baja Verapaz in Guatemala on P. tecunumanii. This clade had high bootstrap support for TEF1, MS204 and RPB2 but was not well supported in the ITS and BT1 gene regions. Three isolates collected from different needles on a single P. tecunumanii tree in Baja Verapaz in Guatemala grouped together in Clade 4 and represent another undescribed species. With the exception of BT1, Clade 4 was statistically well supported in all the gene regions that were analysed.

Clade 5 accommodated sequences representing nine of the 14 known TEF1 haplotypes of L. acicola identified by Janoušek et al. (2016). These TEF1 haplotypes represent isolates collected from North America (Canada, USA, and Mexico), South America (Colombia), Europe (Spain, France, Switzerland, Slovenia, Lithuania, Italy, Germany, Estonia, Czech Republic, Croatia, and Austria) and Asia (South Korea, Japan, and China) (Table 3). This clade was clearly distinct from other clades in the ITS, TEF1, BT1 and MS204 phylogenetic analysis and statistically well supported in the ITS, TEF1, and MS204 analyses. Clade 5 included the ex-type of L. acicola and therefore is that species. None of the isolates from Central America obtained in the present study grouped with this clade in any of the gene regions analysed.

The remaining five assigned L. acicola TEF1 haplotypes considered by Janoušek et al. (2016), grouped together in Clade 6. This was together with an isolate obtained from P. caribaea in Honduras collected in 1983 (Evans 1984), four isolates obtained in the present study from Guatemala on P. oocarpa and P. maximinoi, and an isolate previously identified as L. acicola from Mexico on an unknown Pinus species (Quaedvlieg et al. 2012). In the present study, Clade 6 is treated as a novel taxon. The ITS, TEF1, BT1 and MS204 gene regions clearly distinguish Clades 5 and 6, however, RPB2 was not effective in resolving these two groups.

The second most abundant species collected in this study was Lecanosticta guatemalensis, represented by Clade 7 in the phylogenetic analyses. This clade was well supported in all five gene regions that were analysed. A total of 37 isolates from our collection grouped together with L. guatemalensis based in the ITS and TEF1 analyses. Lecanosticta guatemalensis was identified on P. maximinoi and P. oocarpa in various regions of Guatemala, as well as on P. oocarpa in Nicaragua. Isolates that had previously been collected in Nicaragua and Honduras and that were identified as L. acicola by Evans (1984) based on morphological characteristics also grouped with L. guatemalensis in the present study.

TAXONOMY

Using phylogenetic analyses, 51 of the Lecanosticta isolates obtained from Guatemala, Honduras and Nicaragua, one isolate obtained from CBS, and one isolate obtained from IMI, were found to include four undescribed species. These are described below as follows:

Lecanosticta jani van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826875. (Fig. 3)

Fig. 3

Lecanosticta jani (CMW38958; CMW38950; CMW48831; CMW47109; CMW51058; CMW51143) a-b Two wk. old colonies on MEA. A represents Type 1 colonies (CMW38950) and B represents Type 2 colonies (CMW48831). c-h Conidiogenous cells giving rise to conidia on MEA, with swollen globose basal cells of the conidiophores in E, F and H as well as annelations (see arrow) in G. i-k Swollen conidiogenous cells and conidia on MEA. Note endospore formation and germination in I. l Conidia on MEA. Bars: K = 50 μm; C-F and H-L = 10 μm; G = 5 μm

Etymology: The name is derived from Janus, the Roman god of gates and doorways having two faces or sides, and refers to the variable culture morphology ranging from light pink and fluffy to dark olive green and mucoid.

Diagnosis: Lecanosticta jani can be distinguished from the closely related L. brevispora by the distinct globose basal cells on the conidiophores that are mostly observed on MEA.

Type: Guatemala: Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus oocarpa, 20 Sept 2012, I. Barnes (PREM 62185 – holotype; CMW 38958 = CBS 144456 – ex-type culture).

Description: Sexual morph unknown. Conidiomata isabelline to vinaceous brown on MEA. Conidiophores subcylindrical, often with a swollen globose basal cell, densely aggregated, honey to hyaline, smooth to verruculose, unbranched or branched at base, often encased in a yellow to light brown mucoid sheath, to 82 μm in length, 4.5–7.0 μm diam. Conidiogenous cells terminal, integrated, subcylindrical, honey to hyaline, smooth to verruculose, proliferating several times percurrently with visible annelations near apex, septate or aseptate, (8.5–)16.5(− 24.0) × (3.0–)4.5(− 6.5) μm. Conidia solitary, sub-cylindrical to narrowly fusoid-ellipsoidal, with subobtusely rounded apex, base truncate, brown, verruculose, frequently with mucoid sheath, two distinct sizes with conidial type one more abundant than conidial type two. Conidial type 1: 1–2-septate, base (1.5–)2.0–2.5(− 3.5) μm diam, (9.5–)14.5–21.5(− 30.0) × (2.0–)2.5–3.5(− 4.0) μm. Conidial type 2: 1–3-septate, base (1.5–)2.0–2.5(− 3.0) μm diam, (26.5–)30.5–37.0(− 38.0) × (2.0–)2.5–3.0(− 3.5) μm.

Culture characteristics: Colonies with two distinct morphologies. One type (Type 1), flat to somewhat erumpent, spreading with flat to fluffy aerial mycelium. A second type (Type 2) erumpent, mucoid and shiny, with irregular form and undulate to filiform edges. On MEA, the surface of Type 1 isolates pale to rosy vinaceous, reverse flesh to peach coloured. Type 2 isolates citrine to isabelline, reverse olivaceous to fuscuous black (Fig. 3). On PDA, Type 1 surface rosy vinaceous to peach in centre with dark brown edge, isabelline in reverse. Type 2, surface dark olivaceous with fuscious black centres and tufts of isabelline mycelium at edges, dark isabelline in reverse. On OA, Type 1 surface dirty white to pale vinaceous, fluffy mycelia to flat growth. Type 2 surface flat with smooth edge, fuscious black in centre at the point of inoculation with light apricot surrounding mycelium. Growth characteristics: optimal growth temperature for Type 1 isolates 25 °C, after 4 wk., colonies at 10, 15, 20, 25 and 30 °C reached maximum of 10.5, 22, 32, 32 and 10 mm respectively, with mean growth rate of 2.1, 5.1, 6.9, 7 and 1.8 mm / wk. respectively. Type 2 isolates optimal growth temperature 20 °C, after 4 wk., colonies at 10, 15, 20, 25 and 30 °C reached maximum of 12.5, 17, 29.5, 22 and 4.5 mm, with mean growth of 2.1, 3.3, 5.5, 5 and 1 mm / wk. respectively.

Notes: Lecanosticta jani resolved in a distinct clade (Clade 2, Figs. 1 and 2, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4) based on all five gene regions considered. This clade divides into two subclades that were mostly represented by isolates obtained from Alta Verapaz and Chiquimula in Guatemala as well as in Nicaragua in subclade 1 and isolates obtained from Jalapa in Guatemala in subclade 2. Jalapa isolates all had the Type 2 morphology and the dark colour was associated with conidial production. Type 1 isolates produced few spores after 2 wk. The optimal growth temperature and growth rates were different for the two isolate types. However, the topology of some isolates changed between the two subclades depending on the gene region that is analysed and therefore the subclades are treated as one species. The morphological variation suggests that the two types could represent two ecotypes.

Additional material examined: Guatemala: Alta Verapaz, Santa Cruz Verapaz, near Tactíc, on needles of Pinus oocarpa, 21 Oct 2010, I. Barnes (culture CMW47109); loc. cit. I. Barnes (PREM 62187; CMW 48831 = CBS 144447 – culture); Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus oocarpa, 20 Sept 2012, I. Barnes (PREM 62186, CMW 38950 = CBS 144446 – culture); Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus tecunumanii, 20 Sept 2012, I. Barnes (cultures CMW 51058, CMW 51059). -Nicaragua: Matagalpa, on needles of Pinus oocarpa, 20 June 2011, I. Barnes (culture CMW 51143).

Lecanosticta pharomachri van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826876. (Fig. 4)

Fig. 4

Lecanosticta pharomachri (CMW 37136; CMW38947). a, b Two wk. old colonies on MEA. c-e Conidiogenous cells giving rise to conidia on MEA. f, g Conjugation tube formation between conidia as well as conidia bearing smaller conidial cells. h-j Variation in conidia on MEA. Bars: D, F-H and J = 10 μm; C, E and I = 5 μm

Etymology: The epithet refers to the Resplendid Quetzal (Pharomachrus mocinno), which is the national bird of Guatemala and the spirit bird/companion of Tecún Umán; a Guatemalan legend.

Diagnosis: Lecanosticta pharomachri is distinguished from the other taxa in the genus by all five gene regions investigated but especially by sequences of TEF1, MS204 or RPB2. Conidia are also larger than those of L. guatemalensis and similar to L. acicola but differ from these species in that the conidia are frequently surrounded by a thick mucoid sheath and are mostly straight.

Type: Guatemala: Baja Verapaz, San Jerónimo, Salamá, on needles of Pinus tecunumanii, Nov 2010, I. Barnes (PREM 62188 – holotype; CMW 37136 = CBS 144448 – ex-type cultures).

Description: Sexual morph not observed. Conidiomata dark vinaceous brown on MEA. Conidiophores subcylindrical to cylindrical, densely aggregated, vinaceous brown to hyaline, smooth to verruculose, unbranched or branched at base, often encased in a light brown mucoid sheath, to 45 μm in length, 2.5–4.0 μm diam. Conidiogenous cells terminal, integrated, subcylindrical to cylindrical, luteus brown to hyaline, smooth to verruculose, surrounded by mucilage, holoblastic, proliferating several times percurrently with visible annelations near apex, septate or aseptate, (6.5–)9.5–13.5(− 16.0) × (1.5–)2.0–2.5(− 3.0) μm. Conidia released in a greenish olivaceous to honey mass, solitary, straight to slightly curved, cylindrical, with subobtusely rounded apex, base truncate, guttulate, hyaline to light brown, verruculose, frequently with thick mucoid sheath, 1–3-septate, base (1.5–)2.0–3.0(− 3.5) μm diam, (21.0)25.0–34.0(− 49.0) × (2.5–)3.0–4.0(− 5.0) μm. Germ tubes observed between conidia as well as conidial budding - secondary conidia sometimes produced from apical cell, 0–2-septate.

Culture characteristics: Colonies flat to erumpent, form irregular with undulate edge, spreading with fluffy aerial mycelium at centers. On MEA, surface apricot to cinnamon with isabelline and rosy buff mycelial mat at centers, reverse isabelline to dark brick in centre with cinnamon to apricot edges. On PDA, surfaces rosy to pale vinaceous with light isabelline to greenish white edges, reverse isabelline with cream edges. On OA, surface dirty white to isabelline to dark brown, fluffy mycelium to flat growth. Growth characteristics: optimal growth temperature 20 °C, after 4 wk., colonies at 10, 15, 20, 25, and 30 °C reaching a maximum of 9, 17, 18.5, 18.5 and 8.5 mm diam, with mean growth rates of 1.9, 3.6, 4.6, 4.4, and 1.9 mm / wk. respectively.

Notes: Some of the isolates, including the ex-type strain, produced a luteus exudate that diffused into MEA after 4–6 wk. Conjugation tubes were reported previously in L. acicola cultures as well as in needles (Siggers 1950; Crosby 1966). Conjugation tubes were also observed in this species (Fig. 4g) in the present study. Endospores as described by Crosby (1966) were also observed in some conidia.

Additional material examined: Guatemala: Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus oocarpa, 20 Sept 2012, I. Barnes (cultures CMW 38976, CMW 51053 and CMW 51054); loc. cit., I. Barnes (PREM 62189; CMW 38947 = CBS 144695 – culture; PREM 62190, CMW 38974 = CBS 144449 – culture).

Lecanosticta tecunumanii van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826877. (Fig. 5)

Fig. 5

Lecanosticta tecunumanii (CMW46805; CMW46812). a Two wk. old colony on MEA. b-d Conidiogenous cells giving rise to conidia on MEA. e-f Micronematous conidiogenesis observed on MEA with conidia. g-h Uniseptate conidia with or without a mucoid sheath observed on MEA. Bars: B-G = 10 μm; H = 5 μm

Etymology: Name refers to the Guatemalan legend, Tecún Umán, and Pinus tecunumanii, the host plant from which the holotype was collected.

Diagnosis: Lecanosticta tecunumanii is distinguished from the other taxa by the ITS, TEF1, MS204 and RPB2 gene regions. Morphologically, it is distinct in having only 1-septate conidia after 2 wk. of incubation on MEA, but 2-septate and 3-septate conidia are occasionally observed in older cultures.

Type: Guatemala: Baja Verapaz, San Jerónimo, Salamá, on needles of Pinus tecunumanii, Oct 2011, I. Barnes (PREM 62191 – holotype; CMW 46805 = CBS 144450 – ex-type cultures).

Description: Sexual morph not observed. Conidiomata isabelline to visaceous brown on MEA. Conidiophores cylindrical, densely aggregated, hyaline to pale yellow-brown, smooth to slightly verruculose, unbranched or branched at base, to 120 μm in length, 2.0–5.0 μm diam. Conidiogenous cells terminal or indeterminate, integrated or discrete, cylindrical, hyaline to honey, smooth to verruculose, proliferating several times percurrently with visible annelations near apex or micronematous, septate or aseptate, (5.0–)7.0–14.5(− 15.5) × (1.5–)2.0–2.5(− 3.0) μm. Micronematous cells (6–)10.5–18.5(− 27.0) × (2.0–)2.0–2.5(− 3.0) μm. Conidia solitary, straight to slightly curved, subcylindrical to fusiform, with subobtusely rounded or sharply pointed apex, base truncate, guttulate, smooth to granulate, hyaline to cream buff to light brown, occasionally enclosed in mucoid sheath, 1-septate, base (1.5–)1.5–2.0(− 2.0) μm diam., (14.5–)16.0–21.0(− 24.0) × (2.0–)2.5–3.0(− 3.5) μm.

Culture characteristics: Colonies somewhat erumpent, spreading with flat to fluffy aerial mycelium. On MEA, surface olivaceous to isabelline with rosy buff mycelial tufts, reverse isabelline. On PDA, surface rosy vinaceous to peach in centre with a dark brown edge, isabelline in reverse. On OA, surface dirty white to pale vinaceous, fluffy mycelia to flat peach growth. Growth characteristics: optimal growth temperature 25 °C, after 4 wk., colonies at 10, 15, 20, 25, and 30 °C reached maximum of 9, 15.5, 24, 24, and 4.5 mm, with mean growth of 2.2, 3.8, 5.3, 5.7, and 1.1 mm / wk. respectively.

Notes: Micronematous conidiogenesis (Fig. 5E - F), observed more frequently than distinct conidiophores in culture.

Additional material examined: Guatemala: Baja Verapaz, San Jerónimo, Salamá, on needles of Pinus tecunumanii, Oct 2011, I. Barnes (PREM 62192, CMW 49403 = CBS 144451 – culture; PREM 62193, CMW 46812 = CBS 144452 – culture).

Lecanosticta variabilis van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826878. (Fig. 6)

Fig. 6

Lecanosticta variabilis (CMW42205; CMW37125). a Colony on MEA with luteus exudate diffusing into medium. b-c Conidiogenous cells giving rise to conidia on MEA. d-h Various conidial shapes and sizes on MEA. f Germinating conidia on MEA. g-h Swollen conidial cells giving rise to smaller conidia. i Conjugation tube formation between two conidia. j Conidium disintegrating on MEA. Bars: B-C, F-I = 10 μm; E, J = 5 μm; D = 2,5 μm

Etymology: The epithet refers to the variable size and shape of the conidia.

Diagnosis: Lecanosticta variabilis is distinguished from the closely related species, L. acicola, by either ITS, TEF1 or MS204. Morphologically, it is distinguished from other species with the exception of L. acicola by the diffusion of sulphur-yellow to cinnamon metabolite into PDA and a luteus to sienna coloured metabolite produced on MEA within 2 wk. This species also has smaller conidia than those of L. acicola.

Type: Honduras: Santa Barbara, on needles of Pinus caribaea, 1980, H.C. Evans, (PREM 62196 – holotype; CMW 42205 = IMI 281561 = CBS 144453 – ex-type culture).

Description: Sexual state not observed. Conidiomata olivaceous to vinaceous brown on MEA. Conidiophores cylindrical, extending in densely aggregated palisade, hyaline to honey to pale vinaceous brown, smooth to verruculose, unbranched or branched at base, septate or aseptate, often encased in granular yellow to light brown mucoid sheath, length up to 60 μm, 2.0–5.0 μm diam. Conidiogenous cells terminal, integrated, subcylindrical to cylindrical, hyaline to light brown, smooth to verruculose, proliferating several times percurrently with visible annelations near apex, septate or aseptate, (4.5–)5.5–10.5(− 12.0) × (1.5–)2.0–3.5(− 5.0) μm. Conidia three different conidial types. All three types solitary, smooth to verruculose, subhyaline to honey to light brown, often enclosed in granular light luteus mucoid sheath. Type 1 straight to strongly curved, subcylindrical to cylindrical, subobtusely rounded apex, truncate, 1–4-septate, base (1.5–)2.0–2.5(− 3.0) μm diam. (22–)25.0–34.0(− 43.0) × (2.0–)2.5–3.0(− 3.5) μm. Type 2 slightly curved, cylindrical with both apex and base rounded, 0–2-septate, (14.5–)15.5–19.5(− 22.0) × (2.0–)2.5–3.0(− 3.5) μm. Type 3 buds from larger conidia (see notes) or from conidiogenous cells, hyaline, fusiform to cylindrical with subobtusely rounded apex and base, 0–1-septate, (10.0–)11.0–14.0(− 15.5) × (2.0–)2.0–2.5(− 3.0) μm.

Culture characteristics: Colonies flat to somewhat erumpent, spreading, with sparse aerial mycelium, surface folded, with smooth, lobate margins. On MEA, surface isabelline with patches of pale luteus to dark olivaceous green, reverse olivaceous to fuscous black. Mucoid yellow to peach to yellow-green exudate present. Luteus to sienna coloured metabolite diffusing into medium. On PDA, surface isabelline in centre, rosy buff in outer region, dark olivacous-brown on edges and isabelline in reverse. Sulphur yellow to cinnamon coloured metabolite diffuses into media. On OA, surface dirty white with diffuse umber outer region. Growth characteristics: optimal growth temperature 25 °C, after 4 wk., colonies at 10, 15, 20, 25 and 30 °C reached maximum of 11.5, 21, 31, 31.5 and 22.5 mm, with mean growth of 2.2, 4.5, 6.1, 6.9 and 3.6 mm / wk. respectively.

Notes: The cells in the conidia often swell and break off, forming endospores as described in L. acicola (Siggers 1950; Crosby 1966; Evans 1984). Secondary conidia were commonly produced in cultures of this species, similar to those previously described for L. acicola specimens examined directly from needles (Evans 1984).

Additional material examined: Guatemala: Alta Verapaz, Santa Cruz Verapaz, near Tactíc, on needles of Pinus oocarpa, 21 Oct 2010, I. Barnes (PREM 62194, CMW 37125 = CBS 144454 – culture); loc. cit., I. Barnes (culture CMW 37129); Jalapa, Finca Forestal Soledad, on needles of Pinus maximinoi, 21 Oct 2010, I. Barnes (PREM 62195, CMW 36809 = CBS 144455 – culture). –Mexico: on needles of a Pinus sp., 30 Nov 2009, M. de Jesús Yáñez-Morales (CBS H-21112; culture CMW45425 = CPC 17822 = CBS 133789);

DISCUSSION

Four novel species of Lecanostica from infected pine needles collected in Central America are reported and named as L. jani, L. pharomachri, L. tecunumanii, and L. variabilis. There are now nine species described in the genus and these can be distinguished based on a phylogenetic inference for multiple gene regions. The two previously described species, L. brevispora and L. guatemalensis, were also found in this study and they provide new host and country records. The well-known pine pathogen, L. acicola, was not found on any of the samples collected from five Pinus spp. in seven regions of Central America considered in this study. This suggests that the species is not native in that region.

Results of the present study support the view of Quaedvlieg et al. (2012) that a combination of the ITS and TEF1 should be used as barcoding loci to distinguish between species of Lecanosticta and other closely related species. Additionally, statistically well supported clades were obtained in this study using the MS204 gene region. However, genus-specific primers should ideally be designed to increase the amplification success rate for this gene region in Lecanosticta. Although the BT2 gene was also proposed as a possible barcoding region that could be used to distinguish between Lecanosticta species and other species of Mycosphaerellaceae (Quaedvlieg et al. 2012), it amplified poorly in the present study. The BT1 gene region distinguished most of the species, but not L. pharomachri and L. variabilis and provided low statistical support at all nodes.

The results of this study support the view of Evans (1984) that Lecanostica species are comprised of morphotypes or ecotypes. Based on phylogenetic analyses, we were able to define lineages for species also supported by morphological characteristics. The TEF1 sequences were highly variable but several well supported clades and subclades were observed within species (Fig. 2). These clades possibly represent additional new species but we lacked sufficient cultures and support to describe them. The clade with the most diversity in terms of unique TEF1 haplotypes, Clade 1, was L. brevispora (represented by 22.1% of TEF1 haplotypes in the genus) and this species was also represented by the largest number of isolates. High haplotype diversity was observed in the L. jani (16.1% of TEF1 haplotypes) and L. pharomachri (10.3% of TEF1 haplotypes) clades and different lineages were observed in the L. acicola (13.2% of TEF1 haplotypes), L. guatemalensis (17.6% of TEF1 haplotypes), and L. variabilis (13.2% of TEF1 haplotypes) clades. The other gene regions, especially MS204 and RPB2 were also highly variable in terms of distinguishing haplotypes. RPB2 is however, not recommended to distinguish between L. acicola and L. variabilis as these two species form paraphyletic groups in the tree for this gene region.

The paleo-geographic region that includes Mexico and extends into Central America is regarded as one of three centres of diversity of Pinus species (Farjon 1996). Pine needles that were sampled from Central America in this study were symptomatic but serious disease was not observed. This suggests that Lecanosticta species have co-speciated with their native pine hosts in this region. Of the nine known species, L. gloeospora and L. longispora have been identified only in Mexico and L. brevispora and L. variabilis have been identified in both Mexico and Central America. Lecanosticta guatemalensis, L. jani, L. pharomachri and L. tecunumanii are currently known only from Central America.

Lecanosticta acicola has been redefined in this study. All isolates from Central America that had previously been identified as L. acicola, based on morphological characteristics, are now treated as different species. This is based on newly available DNA sequence data and phylogenetic analyses emerging from this study as well as that of Quaedvlieg et al. (2012). L. acicola is, however, still considered as present in Mexico.

Based on TEF1 analyses, L. acicola resolves in three lineages. Janoušek et al. (2016) used microsatellites to show that a lineage of L. acicola from the northern USA was introduced into Central and Northern Europe, and a lineage from the southern USA was introduced into France, Spain, and Colombia. Similarly, Huang et al. (1995) reported that L. acicola was introduced into China from the southern part of the USA. Our analyses of the TEF1 sequences of isolates from the northern parts of the USA, Lithuania, and a representative sequence for Central and Northern Europe and Canada (KJ938438, Table 3), formed one distinct lineage with L. acicola (Fig. 2). All isolates from the southern parts of the USA, as well as representative sequences for Asia, France, Spain, and Colombia (Table 3), formed a second distinct lineage in the clade accommodating L. acicola (Fig. 2). The third lineage included only isolates from Mexico, which suggests that isolates in this lineage have remained in their area of origin and have not been introduced elsewhere. Because this Mexican lineage had strong bootstrap support separating it from the other two lineages, it could represent a further new species. Only TEF1 data are currently available for the Mexican collections (downloaded from GenBank) and other gene regions would need to be sequenced and analysed to determine whether this really represents a further novel taxon.

Evans (1984) first speculated that Central America could be the centre of origin of Lecanosticta. The phylogenetic analyses conducted in the present study showed that there is a high diversity of species and lineages for this genus in Central America, which supports Evans’ hypothesis. This is the first study where all known species of Lecanosticta have been delineated based on DNA sequence data and phylogenetic analysis, and it has led to the recognition of additional new taxa from Central America and Mexico. Eight of the nine species of Lecanosticta have been reported only from this region, and our results consequently represent strong support for a Mesoamerican Lecanosticta centre of diversity and likely origin. Population genetic analyses for the most common of these species will serve to provide additional support for this hypothesis.

CONCLUSIONS

Phylogenetic inference based on DNA sequence data including new collections from Mexico and Central America revealed four novel species and reaffirmed the identity of the five previously described taxa. The most important of these species is the well-known pine pathogen L. acicola that was redefined as a North American taxon and for which at least three distinct lineages can be distinguished using the TEF1 gene region. New regions of occurrence and host range emerged for Lecanosticta spp. with eight of the nine species occurring in Mesoamerica. This suggests that Mesoamerica is the most likely centre of origin for Lecanosticta. Lecanosticta acicola was best known as the causal agent of the important brown spot needle blight of Pinus palustris in the southeastern USA but it has more recently spread within the USA and Europe where it has become an increasingly important pathogen of numerous Pinus spp. The other species of Lecanosticta, including those newly described, are of unknown importance but it seems likely that some of them could pose a threat to Pinus spp. if they were introduced into new environments in the future. The fact that various Mesoamerican Pinus spp. are increasingly being used for plantation development in the Southern Hemisphere implies that extreme caution should be applied not to introduce Lecanosticta spp. together with germplasm needed for future planting programmes.

Figure S1. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the ITS region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “∆” next to the isolate indicates that the isolate exhibits Type 2 morphology but it groups with Subclade 1 or exhibits Type 1 morphology but groups with Subclade 2. (PPTX 61 kb)

Figure S2. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the BT1 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. (PPTX 54 kb)

Figure S3. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the MS204 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma septosporum was used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “∆” next to the isolate indicates that the isolate exhibits Type 2 morphology but it groups with Subclade 1 or exhibits Type 1 morphology but groups with Subclade 2. (PPTX 55 kb)

Figure S4. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the RPB2 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “∆” next to the isolate indicates that the isolate exhibits Type 2 morphology but it groups with Subclade 1 or exhibits Type 1 morphology but groups with Subclade 2. (PPTX 61 kb)