Reconsideration of species boundaries and proposed DNA barcodes for Calonectria

Abstract

Calonectria represents a genus of phytopathogenic ascomycetous fungi with a worldwide distribution. In recent years, there has been an increase in the number of taxonomic studies on these fungi. Currently, there are 169 described species of Calonectria based on comparisons of DNA sequence data, combined with morphological characteristics. However, for some of these species, the sequence data utilised at the time of their description were relatively limited. This has justified an urgent need to reconsider the species boundaries for Calonectria based on robust genus-wide phylogenetic analyses. In this study, we utilised 240 available isolates including the ex-types of 128 Calonectria species, and re-sequenced eight gene regions ( act , cmdA , his3 , ITS, LSU, rpb2, tef1 and tub2 ) for them. Sequences for 44 Calonectria species, for which cultures could not be obtained, were downloaded from GenBank. DNA sequence data of all the 169 Calonectria species were then used to determine their phylogenetic relationships. As a consequence, 51 species were reduced to synonymy, two new species were identified, and the name Ca. lauri was validated. This resulted in the acceptance of 120 clearly defined Calonectria spp. The overall data revealed that the genus includes 11 species complexes, distributed across the Prolate and Sphaero-Naviculate Groups known to divide Calonectria . The results also made it possible to develop a robust set of DNA barcodes for Calonectria spp. To accomplish this goal, we evaluated the outcomes of each of the eight candidate DNA barcodes for the genus, as well as for each of the 11 species complexes. No single gene region provided a clear identity for all Calonectria species. Sequences of the tef1 and tub2 genes were the most reliable markers; those for the cmdA , his3 , rpb2 and act gene regions also provided a relatively effective resolution for Calonectria spp., while the ITS and LSU failed to produce useful barcodes for species discrimination. At the species complex level, results showed that the most informative barcodes were inconsistent, but that a combination of six candidate barcodes ( tef1 , tub2 , cmdA , his3 , rpb2 and act ) provided stable and reliable resolution for all 11 species complexes. A six-gene combined phylogeny resolved all 120 Calonectria species, and revealed that tef1 , ...

Species Diversity, Mating Strategy and Pathogenicity of Calonectria Species from Diseased Leaves and Soils in the Eucalyptus Plantation in Southern China

“…According to previous research results, sequences of partial gene regions of translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) were used to successfully identify Calonectria species [26,28,32]. These four partial gene regions were amplified using the primer pairs EF1-728F/EF2, T1/CYLTUB1R, CAL-228F/CAL-2Rd and CYLH3F/CYLH3R, respectively, the PCR procedure was conducted as described by Liu and Chen [33], Lombard and co-authors [30] (Table 1).…”

“…Sequences of all the published species in the relative species complexes were used for sequence comparisons and phylogenetic analyses. The datasets of Liu and co-authors [28] were used as templates for analyses.…”

“…Maximum parsimony (MP) and maximum likelihood (ML) were used frequently for phylogenetic analyses of Calonectria species [28,29,36]. To test whether the analysis results between the two methods are consistent, both MP and ML were used for phylogenetic analyses for sequence datasets of each of the four genes and the combination of four gene regions.…”

“…Phylogenetic trees were viewed using MEGA v. 6.0.5 [34]. Sequence data of two isolates of Curvicladiella cignea (CBS 109167 and CBS 109168) were used as outgroups [28].…”

“…Results of the standard nucleotide BLAST search conducted using the tef1, tub2, cmdA and his3 sequences showed that the isolates obtained in the current study belong three species complexes of Calonectria, including C. reteaudii species complex, the C. cylindrospora species complex, and the C. kyotensis species complex. Based on the recently published results in Liu and co-authors [28], sequences of tef1, tub2, cmdA and his3 published species in the C. reteaudii species complex, C. cylindrospora species complex and C. kyotensis species complex, respectively, were used for sequence comparisons and phylogenetic analyses (Appendix A Table A2).…”