Diversity and Distribution of Calonectria Species from Plantation and Forest Soils in Fujian Province, China

To meet the growing demand for wood and pulp products, Eucalyptus plantations have expanded rapidly during the past two decades, becoming an integral part of the southern China landscape. Leaf blight caused by various Calonectria spp., is a serious threat to these plantations. In order to explore the diversity and distribution of Calonectria spp. in Fujian Province soils, samples were collected in Eucalyptus plantations and adjacent plantings of Cunninghamia lanceolata, Phyllostachys heterocycle and Pinus massoniana as well as in natural forests. Three hundred and fifty-three Calonectria isolates were recovered from soil samples and they were identified based on a comparison of multilocus DNA sequence data for the act (actin), cmdA (calmodulin), his3 (histone H3), rpb2 (the second largest subunit of RNA polymerase), tef1 (translation elongation factor 1-alpha) and tub2 (β-tubulin) gene regions, as well as morphological characteristics. Six known taxa including Calonectria aconidialis, Ca. hongkongensis, Ca. ilicicola, Ca. kyotensis, Ca. pacifica, Ca. pseudoreteaudii and one novel species described here as Ca. minensis sp. nov. were identified. Of these, Ca. aconidialis and Ca. kyotensis were the most prevalent species, and found in eight and seven sites, and four and five forest types, respectively. Calonectria spp. were most abundant in soils from Eucalyptus stands, followed by P. heterocycle and natural forests. Relatively few species were found in the soils associated with Cunninghamia lanceolata and Pinus massoniana. The abundance of known Calonectria spp. suggests that these fungi have been relatively well sampled in Fujian. The results are also consistent with the fact that most Calonectria diseases are found on Angiosperm as opposed to Gymnosperm plants.


Introduction
Species of Eucalyptus are the most important trees used to establish plantations in the tropics and Southern Hemisphere, where they provide substantial resources for the global fibre market [1]. These trees were first introduced into China as ornamentals in 1890 and plantations of Eucalyptus spp. had reached 5.46 million hm 2 by 2018 [1]. Plantations of these trees are mainly distributed in 11 provinces of China, and over 75% can be found in the Guangxi, Guangdong, Yunnan and Fujian Provinces of southern China [1]. The Eucalyptus plantations in China have been established with a relatively narrow genetic base and consequently many disease problems, caused by a variety of pathogens, have emerged as threats to their sustainability [2][3][4][5][6].
Among the diseases threatening Eucalyptus plantations, leaf blight caused by species of Calonectria De Not. has become a major constraint in southern China [4,[7][8][9][10]. Symptoms of infection are characterised by water-soaked spots on leaves in the lower and middle parts of the tree crowns. These coalesce and gradually develop into extended necrotic areas, which result in blight and often severe defoliation [9]. In China, Calonectria Leaf Blight (CLB) has been observed in Eucalyptus plantations in Fujian, Guangdong, Guangxi, Hainan and Yunnan Provinces [4,7,[9][10][11]. This is similar to the situation in Australia, Brazil, Indonesia, Thailand and Vietnam where Eucalyptus plantations have also suffered significant damage due to CLB [12][13][14][15][16].
The genus Calonectria includes many aggressive plant pathogens. These species are extensively distributed particularly in sub-tropical and tropical regions of the world, and they have a wide host range including more than 335 plant species [17]. Calonectria species are generally considered as soil-borne fungi and they can survive in the soil for extended periods due to their thick-walled microsclerotia [17].
Plantations of Eucalyptus spp. are commonly established alongside those of Cunninghamia lanceolata, Phyllostachys heterocycle and Pinus massoniana and can also be in mixed plantings in the Fujian Province ( Figure 1). In recent years, leaf blight has become a serious threat to Eucalyptus plantations in this province [7,8]. Calonectria species including Ca. crousiana, Ca. eucalypti, Ca. fujianensis, Ca. pauciromosa and Ca. pseudoreteaudii [7,8,18] have been isolated from diseased Eucalyptus tissues and are regarded as the important causal agents of CLB in Fujian. Calonectria infections initially arise from inoculum in the soil but very little is known regarding the species diversity and distribution of these fungi in Fujian soils. The aim of this study was thus to determine the identity and distribution of Calonectria spp. from a wide variety of soils in Fujian, with a particular focus on Eucalyptus spp. but also including other trees that are found in the area.  Ca. aconidialis (12); Ca. kyotensis (8); Ca. pacifica (8) f
The PCR reaction mixtures contained 17.5 µL TopTaq TM Master Mix, 1 µL of each primer (10 mM), 2 µL DNA sample and RNase-Free H 2 O to a final volume of 35 µL. The amplifications were conducted under conditions described by Liu and co-authors [18]. All PCR products were sequenced in both directions using the same primers used for amplification. Raw sequences were inspected and manually corrected in Geneious v. 9.1.4 (Biomatters, Auckland, New Zealand) [28]. All sequences generated in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov; accessed on 24 July 2022) ( Table 2, Appendix A Table A1).

Phylogenetic Analyses
To obtain the preliminary identification of the isolates, a standard nucleotide BLAST search was conducted using sequences of the six (act, cmdA, his3, rpb2, tef1 and tub2) gene regions. Furthermore, sequences obtained in this study ( Table 2) and sequences of other phylogenetically closely related Calonectria species downloaded from NCBI (http: //www.ncbi.nlm.nih.gov; accessed on 24 July 2022) ( Table 3) were used in the analyses. Sequence alignments were conducted online with MAFFT v. 7 (Suita, Janpan) [29] and were manually adjusted in MEGA v. 6.0.5 software (Auckland, New Zealand) [30] when necessary. The final alignments used in phylogenetic analyses were submitted to TreeBASE (http://treebase.org; accessed on 3 October 2021).
Genotypes of all the isolates were determined based on the sequences for the six gene regions. Representative isolates for all the genotypes were selected for the phylogenetic analyses. All the isolates of the novel species were used in the analyses. Maximum Parsimony (MP) and Maximum Likelihood (ML) approaches were used for phylogenetic analyses. The sequence datasets for the six individual gene regions and a concatenated dataset for those regions were used to determine the phylogenetic relatedness of all the isolates. PAUP v. 4.0 b10 [31] was used to perform the MP analyses, and PhyML v. 3.0 [32] was applied to conduct the ML analyses. A partition homogeneity test (PHT) [33] was performed to assess whether the datasets for the six gene regions could be combined.
For MP analyses, all characters were unordered and equally weighted. Gaps were regarded as fifth character and phylogenetic trees were obtained using a heuristic tree search criterion including 1000 random stepwise additions and tree-bisection-reconstruction (TBR) branch swapping. Branches of zero-length were collapsed. Supports for tree-branching points were determined using bootstrap analyses with 1000 replicates [34]. Tree length (TL), retention index (RI), consistency index (CI), rescaled consistency indexes (RC) and homoplasy index (HI) ( Table 4) were calculated for parsimony trees. For ML analyses, the best substitution model for each dataset was determined using JModeltest 2.1.7 [35]. Sequence data for two isolates of Curvicladiella cignea (CBS 109167 and CBS 109168) were used as outgroup taxa (Table 3).  Microbiological Culture Collection Center, Beijing, China. c Isolates used in phylogenetic analyses. d Genotype within each identified species, determined by sequences of act, cmdA, his3, rpb2, tef1 and tub2 regions; '-' means not available. e act = actin; cmdA = calmodulin; his3 = histone H3; rpb2 = the second largest subunit of RNA polymerase; tef1 = translation elongation factor 1-alpha; tub2 = β-tubulin. f N/A represents sequences that are not available. g Isolates used in morphological and culture growth studies. h Isolates used for mating studies. i Isolates that represent ex-type cultures are indicated in bold.

Sexual Compatibility
The mating system as either homothallic or heterothallic was determined for the novel species identified in this study. Representative isolates of this species were crossed with each other in all possible combinations. These crosses were made on minimum salt agar (MSA) [54] with autoclaved toothpicks randomly placed on the agar surface. Petri dishes were then incubated at 25 • C for 2-8 wk, and they were observed regularly for the appearance of perithecia. When perithecia extruding ascospores emerged, germination tests were conducted to determine if the spores were viable. Production of viable ascospores was accepted as an indication of successful mating.

Morphology
Representative isolates of the novel species identified in this study were selected for morphological characterisation. Synthetic nutrient-poor agar (SNA) [55] was used to induce the asexual morphs. Agar plugs from axenic cultures were transferred to SNA and incubated at 25 • C for seven days. Fungal structures were lifted from the plates using a sterile needle and transferred to a drop of 85% lactic acid on microscope slides. Microscopic structures were examined under a Zeiss Axio Imager A1 microscope (Carl Zeiss Ltd., Jena, Germany).
In the case of sexual structures, the perithecia were transferred to Jung tissue freezing medium (Leica Biosystems, Wetzlar, Germany), which was frozen at −20 • C for ten minutes. Vertical sections (10 µm thick) were cut through the perithecia on a HM550 cryostat microtome (Microme International GmbH, Termo Fisher Scientifc, Walldorf, Germany) at −20 • C and examined under an Axio Imager A1 microscope.
For cultures selected as the ex-type isolates, 50 replicate measurements were made for each taxonomically characteristic structure. For other isolates, 30 replicate measurements were made. Minimum, maximum and average (mean) measurements were recorded as (minimum-) (average-standard deviation)-(average + standard deviation) (-maximum).
Optimal growth temperatures for the novel species were determined on MEA. Agar plugs were removed from the actively growing edges of 7-day-old cultures with a 5 mm diam. cork borer and transferred to the centres of 90 mm Petri dishes containing MEA. Cultures were grown at seven different temperatures ranging from 5 • C to 35 • C, at 5 • C intervals with five replicates per isolate. Colony diameters were measured after seven days. Colony colours were described using the colour charts of Rayner [56] using seven-dayold cultures on MEA incubated at 25 • C. All descriptions were deposited in MycoBank (www.mycobank.org, accessed on 3 October 2021).

Sample Collection and Fungal Isolation
A total of 209 soil samples were collected and 353 isolates having a morphology typical of Calonectria were isolated from 79 of these samples (Table 1, Appendix A Table A1). Of these, 121 soil samples were from seven Eucalyptus plantations, of which 57 samples yielded 253 Calonectria isolates. Forty-three soil samples were collected from four natural forests, of which 14 samples yielded 61 Calonectria isolates; 21 soil samples were collected from two C. lanceolata plantations, two of which yielded nine Calonectria isolates; and 14 soil samples collected from a single P. heterocycle plantation, of which five samples yielded 25 Calonectria isolates. In addition, ten soil samples were collected from the Pi. massoniana plantation, only one of which yielded five Calonectria isolates (Table 1).

Phylogenetic Analyses
The tef1 fragment was amplified for all of the 353 isolates (Appendix A Table A1), and based on sequence differences for this region and the sampling sites, 144 isolates were selected to amplify the cmdA, his3 and tub2 gene regions. Subsequently, based on the 37 genotypes revealed by these four gene regions, 71 representative isolates were chosen to amplify the act and rpb2 gene regions (Appendix A Table A1). All of the 71 isolates, representing the 40 genotypes determined from the sequence data for the six gene regions, were used for phylogenetic inference (Table 2). Amplicons generated for the act, cmdA, his3, rpb2, tef1, and tub2 gene regions were approximately 300, 700, 500, 860, 550, and 600 bp, respectively.
Sequence data for 46 Calonectria species closely related to those collected in this study were downloaded from GenBank and a total of 78 sequences (for ex-type and other strains) from previous studies were included in the phylogenetic analyses (Table 3). Phylogenetic analyses based on the six individual gene regions and the concatenated dataset for those regions were conducted using both MP and ML methods. The results showed that the overall topologies generated from the MP analyses were essentially similar to those from the ML analyses, and consequently, only the ML trees are presented (   . Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of combined DNA dataset of act, cmdA, his3, rpb2, tef1, and tub2 gene sequences. Bootstrap value ≥70% for ML and MP analyses are presented above the branches. Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168).
The partition homogeneity test carried out on the datasets, for the combined six gene regions, generated p values of 0.001. This showed that the accuracy of the combined data did not suffer relative to the individual partitions [57]. Sequence data for the six gene regions were thus combined for analyses. The sequence alignments based on the individual six gene regions and the combination of these were deposited in TreeBASE (No. S28845). Statistics and important parameters emerging from the phylogenetic analyses are presented in Table 4.
Based on the six-gene combined phylogenetic tree (Figure 3), for the 71 isolates used in the phylogenetic analyses, eight isolates resided in the Ca. colhounii species complex, two isolates in the Ca. reteaudii species complex and 61 isolates in the Ca. kyotensis species complex.

Species in the Calonectria colhounii Species Complex
Six isolates (CSF9941, CSF9974, CSF9975, CSF9976, CSF9977 and CSF9978), representing one genotype, formed a distinct lineage in the cmdA and tub2 analyses as well as in the six-gene combined phylogenetic tree (Figure 3, Appendix B Figures A2 and A6). The total number of SNP differences between the six isolates and other phylogenetically closely related species [Ca. aciculata (ex-type isolate CERC 5342), Ca. colhounii (ex-type isolate CBS 293.79), Ca. eucalypti (ex-type isolate CMW 18444) and Ca. honghensis (ex-type isolate CERC 5572)] for six gene regions combined, varied between 13 and 31. Thus, this fungus can be regarded as a novel species. Two isolates (CSF9933 and CSF9934) formed an independent clade and were phylogenetically most closely related to the six isolates in the six-gene phylogenetic tree (Figure 3). These two isolates were consequently considered as the same species as the six isolates CSF9941, CSF9974, CSF9975, CSF9976, CSF9977 and CSF9978 and were identified as the novel species.

Species in the Calonectria reteaudii Species Complex
Two isolates (CSF10059 and CSF10060) were phylogenetically closely related to Ca. pseudoreteaudii and various other species based on act and cmdA trees (Appendix B Figures A1 and A2), and clustered with Ca. pseudoreteaudii based on his3, rpb2, tef1, tub2 and the six-gene combined trees (Figure 3, Appendix B Figures A3-A6). In comparisons of DNA sequences for these six gene regions, all the sequences for the two isolates (CSF10059 and CSF10060) were 100% identical to the ex-type isolate (CMW 25310) of Ca. pseudoreteaudii. Consequently, they were identified as Ca. pseudoreteaudii (Figure 3).

Species in the Calonectria kyotensis Species Complex
Thirty-four isolates representing 20 genotypes were phylogenetically closest to Ca. kyotensis in each of the cmdA, his3, rpb2 (sequence data for the rpb2 were not available for isolate CSF9834), tef1, tub2 and the six-gene combined trees (Figure 3, Appendix B Figures A2-A6), and clustered with Ca. kyotensis based on the act tree (Appendix B Figure A1). Some isolates formed distinct clades based on the six-gene combined trees (Figure 3), while the total number of SNP differences between the 34 isolates and the extype isolate of Ca. kyotensis (CBS 114525) for six gene regions combined varied between 2 and 8. Based on the phylogenetic analyses, these 34 isolates were identified as Ca. kyotensis.
Four isolates (CSF7124, CSF9784, CSF9794 and CSF9799), representing two genotypes, were phylogenetically closest to Ca. hongkongensis in each of the cmdA, tub2 and six-gene combined tree (Figure 3, Appendix B Figures A2 and A6), and clustered with Ca. hongkongensis based on act, his3, rpb2 and tef1 trees (Appendix B Figures A1 and A3-A5). There were only three or four SNP differences between these four isolates and the ex-type isolate of Ca. hongkongensis (CBS 114828) when sequences for six gene regions were combined. Thus, these four isolates were identified as Ca. hongkongensis.
Two isolates (CSF9862 and CSF9863), representing one genotype clustered with Ca. ilicicola in the his3 tree (Appendix B Figure A3), formed independent clades but closely related to Ca. ilicicola in the act, cmdA, rpb2, tef1 and six-gene combined trees (Figure 3, Appendix B Figures A1, A2 and A4-A6). There were only six SNP differences between the two isolates and the ex-type isolate of Ca. ilicicola (CMW 30998) for five gene regions (tub2 sequence data were not available for Ca. ilicicola) combined. Consequently, these isolates were regarded as Ca. ilicicola.
Four isolates (CSF10024, CSF10070, CSF10077 and CSF10129), representing three genotypes, were phylogenetically related to Ca. pacifica and various other closely related species based on act and tef1 trees (Appendix B Figures A1 and A5). They were, however, phylogenetically closest to Ca. pacifica based on his3 and six-gene combined trees (Appendix B Figure A3), and clustered with Ca. pacifica based on cmdA and rpb2 trees (Appendix B Figures A2 and A4). There were only one or three SNP difference(s) between the four isolates and the ex-type isolate of Ca. pacifica (CMW 16726) for five gene regions (tub2 sequence data were not available for Ca. pacifica) combined. These four isolates were thus identified as Ca. pacifica.
Seventeen isolates representing 11 genotypes were phylogenetically closest to Ca. aconidialis based on cmdA, his3, tef1 and six-gene combined trees (Figure 3, Appendix B Figures A2, A3 and A5), and clustered with Ca. aconidialis based on act and rpb2 (rpb2 sequence data were not available for CSF9779 and CSF9875) trees (Appendix B Figures A1 and A4). Some isolates formed distinct clades based on the six-gene combined trees (Figure 3), while the total number of SNP differences between the 17 isolates and the ex-type isolate of Ca. aconidialis (CMW 35174) for five gene regions (sequence data for the tub2 region were not available for Ca. aconidialis) combined varied between 0 and 4. Therefore, the 17 isolates were identified as Ca. aconidialis.
Seventy-one of the 353 isolates collected in this study were identified based on the DNA sequence of the six gene regions. According to the species identification results, we further identified the remaining 282 isolates based on the DNA sequences for two or four gene regions (Appendix A Table A1). Consequently, for the entire collection of 353 isolates, these were identified as Ca. aconidialis (178), Ca. kyotensis (103), Ca. hongkongensis (37), Ca. pacifica (17), Ca. ilicicola (five), Ca. pseudoreteaudii (five) and a novel species (eight), respectively.

Sexual Compatibility
Three isolates (CSF9933, CSF9941 and CSF9975) of the novel species were used in the mating tests ( Table 2). All of these isolates formed protoperithecia readily within two weeks, and perithecia with viable ascospores were produced within four weeks. This was irrespective of whether they were crossed with each other or with themselves. The species was thus shown to be homothallic.

Morphology and Taxonomy
Based on multi-gene phylogenetic analyses (Figure 3, Appendix B Figures A1-A6) and morphological characteristics, seven Calonectria species were identified in this study, including six described species, i.e., Ca. aconidialis, Ca. kyotensis, Ca. hongkongensis, Ca. pacifica, Ca. ilicicola, Ca. pseudoreteaudii and one novel species. To facilitate future studies, complete morphological descriptions and illustrations have been made for the known species and these are presented in Appendix C ( Figures A7-A12). The novel species can be distinguished from the phylogenetically most closely related species (Ca. aciculata, Ca. colhounii, Ca. eucalypti and Ca. honghensis) by the dimensions of its macroconidia and ascospores (Table 5). This species is described as follows: Table 5. Morphological comparisons of Calonectria species obtained in this study and other phylogenetically closely related species. Ca. aciculata

Distribution of Calonectria Species in Fujian Province
Of the seven Calonectria species identified, Ca. aconidialis accounted for 50.4% of all the isolates. This was followed in order of occurrence by Ca. kyotensis (29.2%), Ca. hongkongensis  Between two and four Calonectria species were isolated from soils sampled at each of the nine Counties or Districts (Figure 2). Calonectria aconidialis was found at all sites other than Cangshan District, Ca. kyotensis was found at all sites other than Yanping District and Zhangping County, and the remaining five species were found at between one and three sampling sites (Figure 2).
All seven species were isolated from soils collected in Eucalyptus plantations. Five of the species were isolated from soils in natural forests, the exception being Ca. ilicicola and Ca. pesudoreteaudii. Only Ca. aconidialis and Ca. kyotensis were isolated from soils in P. heterocycle and C. lanceolata plantations, and only Ca. kyotensis was collected from soils in the Pi. massoniana plantation ( Figure 5). Based on the percentage of soil samples that obtained Calonectria from each of the five forest types, the results showed that Calonectria was widely distributed in Eucalyptus plantation soils (47.1%, 57 of 121 sampled soils), followed by P. heterocycle (35.7%, 5 of 14 sampled soils) and natural forests (32.6%, 14 of 43 sampled soils), only 10% of soil samples obtained Calonectria from C. lanceolata (2 of 21 sampled soils) or Pi. massoniana (1 of 10 sampled soils).
Calonectria kyotensis was detected in soils in all of the soil types sampled, while Ca. aconidialis was isolated from soils in all forest types other than Pi. massoniana. Calonectria hongkongensis, Ca. pacifica and Ca. minensis were found both in Eucalyptus plantations and natural forests and the remaining two species were found only in Eucalyptus plantations ( Figure 5).

Discussion
A total of 353 Calonectria isolates were collected from soils in Eucalyptus plantations and adjacent plantations of other species or natural forests in Fujian Province. Multilocus phylogenetic inference and morphological characteristics revealed seven Calonectria species including Ca. aconidialis, Ca. hongkongensis, Ca. ilicicola, Ca. kyotensis, Ca. pacifica and Ca. pseudoreteaudii, and a novel species described here as Ca. minensis.
Results in this study showed that Ca. aconidialis and Ca. kyotensis were the most prevalent species in the soils sampled. Calonectria aconidialis accounted for 50.4% of all the isolates, which was found in eight of the nine sampled sites and soils of all forest types other than those of Pi. massoniana. The next most common species was Ca. kyotensis, accounting for 29.2% of the isolates, which was isolated from seven sites and soils of all five different forest types. The remaining five species were less common, and isolated only from one to three sites, either from Eucalyptus plantations or natural forests, or from both of these forest types.
Among the identified species, Ca. aconidialis is newly reported in Fujian Province and Ca. pacifica represents a first record for China. Eight Calonectria species were previously known in Fujian Province. These include Ca. crousiana, Ca. eucalypti, Ca. fujianensis, Ca. pauciramosa and Ca. pseudoreteaudii collected from diseased Eucalyptus leaves [7,8], Ca. hongkongensis and Ca. kyotensis isolated from soils in unknown forest types [4,18] and Ca. ilicicola collected from diseased peanuts (Arachis hypogaea) in Longyan Region [58].
The Calonectria species diversity in soils was clearly dependent on the forest types sampled. Of the seven species detected, all were obtained from Eucalyptus plantations, five were obtained from natural forests and only one or two species were from other forest types. While these observations are convincing in terms of broad patterns, they must be tempered by the fact that the greatest number of soil samples were from Eucalyptus plantations and natural forests, which could have influenced the results.
Five species residing in the Ca. kyotensis species complex were identified in the present study. Of these, Ca. aconidialis accounted for more than half of all the isolates collected, and has previously been shown to be widely distributed in soils of Eucalyptus plantation in many regions of southern China, including Guangdong [11,18], Guangxi [4,10,11] and Hainan Provinces [11]. In the present study, Ca. aconidialis was collected from soils of four types of forests and in eight of the nine sampling sites in Fujian Province (Figure 2), providing new geographic records for this pathogen in China. This species has previously been shown to infect inoculated Eucalyptus seedlings [10] and could pose a threat to Eucalyptus plantation forestry. Calonectria pacifica was isolated from soils both in the Eucalyptus plantations (Minhou and Yongan Counties) and natural forests (Yanping District) in this study. This species was originally described on Araucaria heterophylla from Hawaii, USA [40], and this is the first report of the fungus in China.
This study elucidated the diversity and distribution characteristics of Calonectria species in soils collected from plantations and natural forests in Fujian Province. Broad patterns of occurrence were clear with Eucalyptus soils yielding the largest number of species. The conifer forests had the lowest number of species, which is consistent with the fact that most Calonectria spp. are known from Angiosperm hosts or from soils associated with these plants. The results of the present study bring the number of Calonectria species recorded in Fujian to 11. Most of these species have also been shown to be pathogenic to Eucalyptus in previous studies [7,9,10]. The surprisingly high species diversity in this region suggests that Calonectria species will pose long-term challenges for the development of Eucalyptus forestry in southern China.

Data Availability Statement:
The sequences from the current study were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 24 July 2022) and the accession numbers were listed in Table 2.     ; rpb2 = the second largest subunit of RNA polymerase; tef1 = translation elongation factor 1-alpha; tub2 = β-tubulin. e Isolates used in phylogenetic analyses. f N/A represents the relative locus was not successfully amplified in the current study. g '-' represents the relative locus was not amplified in the current study. h Isolates used in morphological and culture growth studies. i Isolates used for mating studies. j Isolates that represent ex-type cultures are indicated in bold.

Appendix B. Phylogenetic Tree of Calonectria Species Based on Maximum Likelihood
(ML) Analyses of act, cmdA, his3, rpb2, tef1 and tub2 Gene Sequences Figure A1. Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of act gene sequences. Bootstrap value ≥70% for ML and MP analyses are presented at the branches. Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168). Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168). Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168). Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168). Figure A5. Phylogenetic tree of Calonectria species based on maximum likelihood (ML) analyses of tef1 gene sequences. Bootstrap value ≥70% for ML and MP analyses are presented at the branches. Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168). Bootstrap values lower than 70% are marked with "*", and absent analyses values are marked with "-". Ex-type isolates are marked with "T". Isolates sequenced in this study are highlighted in blue and bold type. The "B" species codes are consistent with the recently published results in Liu and co-authors [18]. The tree was rooted to Curvicladiella cignea (CBS 109167 and CBS 109168). Description: Ascomata perithecial, solitary or in groups of up to three, orange, becoming red-brown with age; in section, apex and body orange, base dark red-brown, subglobose to ovoid, 243-376 µm high, 219-355 µm diam, body turning red, and base dark red-brown in 3% KOH+; ascomatal wall rough, consisting of two thick-walled layers; outer layer of textura globulosa, 31-54 µm thick, cells becoming more compressed towards the inner layer of textura angularis, 10-28 µm thick, cells becoming thin-walled and hyaline towards the centre; outermost cells 10-25 × 9-23 µm, cells of inner layer 6-24 × 2-6 µm; ascomatal