A GMYC analysis was run in the single threshold mode (Figure 4.4) and the result of this analysis delimited the specimens into five putative species (ML entities) but with a confidence interval range from 4 to 365. The phylogenetic tree was analysed by GMYC using the multiple thresholds mode as well to account for the fact that coalescent depths among species are different. The result of this analysis (Figure 4.5) delimited the specimens into 34 putative species with a confidence interval range from 23 to 184.
Figure 4.4 Lineage-through-time (LTT) plot (left) and phylogenetic tree analysed based on the estimated threshold (right) investigated for mycobiont of Usnea specimens using General Mixed-Yule Coalescent approach in the single threshold mode. The sudden rise in branching rate was considered to be the transition from interspecific to intraspecific region (red line) and the delimited lineages based on this line were shown using the red branches on the phylogenetic tree.
Figure 4.5 Lineage-through-time (LTT) plot (left) and phylogenetic tree analysed based on the estimated thresholds (right) investigated for mycobiont of Usnea specimens using General Mixed-Yule Coalescent approach in the multiple thresholds mode. The sudden rise in branching rate was considered as the transition from interspecific to intraspecific region (red lines) and the delimited lineages were shown using different colours in phylogenetic tree.
After labelling the specimens that were identified by experts on the phylogenetic tree, several well- supported clades were formed (Figure 4.6). Most of the identified specimens were placed in distinct species clades. However, one of the U. torulosa specimens (81a) was placed in the U. inermis clade, and one of the U. rubicunda specimens (5425) was completely separated from the other two and was placed on a different clade. Therefore, based on the ITS rDNA phylogenetic positions, these two specimens (81a and 5425) might have been misidentified. However, it seems that ITS rDNA region of
U. torulosa is very similar to that of U. inermis because the other identified U. torulosa specimen is the
closest species to the U. inermis clade. For four species, U. angulata, U. ciliata, U. cornuta, and U.
xanthopoga, there was only one specimen per species; however, they were separated on distinct
clades. Based on the Usnea phylogenetic topology, U. cornuta, U. rubicunda, U. angulata, and U.
articulata among the identified specimens in this study should be present in the Usnea distinct clade
and U. ciliata belongs to the Neuropogon clade. Therefore, a different classification compared to Figure 4.2 was carried out based on this topology to visualise four clades of Usnea, Neuropogon,
U. xanthopoga, and U. ciliifera reported to be a part of Usnea clade while U. nidifica is suggested to
be a member of the Eumitria clade.
Figure 4.6 Identified specimens of Usnea on the mycobiont phylogenetic tree. Some well- supported species clades were formed: U. rubicunda (orange clade), U. inermis (grey clade), U. ciliifera (red clade), U. articulata (green clade), and U. nidifica (light blue clade). Based on the previously reported Usnea phylogenetic topology (Truong et al., 2013; Wirtz et al., 2006) four distinct groups of Usnea, Neuropogon, DolichoUsnea, and
Eumitiria were estimated and labelled on this tree by considering the available species. Except U. ciliata which belongs to Neuropogon clade and U. nidifica which belongs to
Eumitria clade, the rest of identified specimens placed in the Usnea clade. No identified sample was placed in the DolichoUsnea clade.
A few well-supported clades were formed after labelling the tree with the results from the sequence similarity BLAST search given in Table 4.6 (Figure 4.7). Except for the specimens that were similar to
U. cornuta, which were placed in two different clades, all other species (U. ciliata, U. dasaea, U. esperantiana, and U. rubrotincta) formed their own distinct clades. However, the majority of
specimens that were similar to U. cornuta (592, 593, 594, 595, and 604) were placed in the clade which was supported by the only morphologically identified U. cornuta (CORN). Some disagreements were
found by comparing these well-supported clades with the clades formed based on the morphologically identified specimens (Figure 4.6) because the U. dasaea distinct clade included the specimen 5423, which was identified as U. rubicunda morphologically. Based on the Usnea phylogenetic topology and the previous classification (Figure 4.6), the specimens MAX1, N11, 547, and 548 cannot be U. cornuta because they were placed in DolichoUsnea clade.
Figure 4.7 Mycobiont phylogenetic tree containing the sequence-similarity BLAST search results. Specimens similar to U. cornuta produced two separated clades but the other specimens similar to U. esperantiana, U. rubtotincta, and U. dasaea formed only one well-supported clade for each species.
Figure 4.8 shows the mycobiont phylogenetic tree that was constructed using the ITS rDNA of 45 species of Usnea from GenBank and the DNA sequences produced in this study. Some of these species formed distinct clades. The presence of only a few species of these 45 species was suggested based
U. esperantiana, U. rubrotincta, U. subcapillaris, and U. subdasaea. Analysis of this phylogenetic tree
using GMYC analysis in the single mode (Figure 4.9) delimited the species in to 99 distinct species clade (confidence interval: 12-113 species). However, several poorly estimated clades were observed and different species were considered to be one species. For example, U. subfloridana and U. florida, U.
glabrescens and U. fulvoreagens, U. subantarctica and U. trachycarpa, and U. antarctica and U. aurantiacoatra were considered to be the same species. Analysing the same phylogenetic tree using
GMYC in the multiple mode (Figure 4.10) increased the suggested 135 delimited species (confidence interval: 64-195 species). By looking at the GMYC analysis result, it was obvious that there were still several misestimated distinct clusters among the distinct clusters suggested by multiple thresholds. For example, U. subfloridana and U. florida considered to be one distinct species, as were U. antarctica and U. aurantiacoatra. For example, U. trichodeoides was separated from U. diffracta, whereas these two species belong to the DolichoUsnea clade based on the Usnea phylogenetic topology. By looking at the topology of this constructed tree, specimens belonging to the Neuropogon group formed two separated clades (labelled in Figure 4.8); one included U. ciliata and U. subcapillaris, and the other one included U. patagonica, U. antarctica, U. acromelana, U. lambii, U. perpusilla, U. ushuaiensis, U.
subantarctica, U. trachycarpa, and U. sphacelata which is similar to the reported topology for Usnea
in the phylogenetic literature. None of the ITS rDNA sequences generated from three specimens identified as U. rubicunda (5423, 5424, and 5425) produced a clade with the U. rubicunda ITS rDNA collected from GenBank (JQ837314); they were placed in the U. dasaea, U. subdasaea, and U.
rubrotincta clades, respectively. The three specimens that were morphologically identified as U. articulata and used in this study (204, AK6, and NV1) formed a clade together but the U. articulata ITS
Figure 4.8 The Usnea phylogenetic tree constructed using the ITS rDNA sequences of 45 different species of Usnea collected from GenBank and ITS rDNA sequences from 367 New Zealand Usnea specimens. U. subcapillaris, U. ciliata, U. dasaea, U. angulata, U. subdasaea, U. esperantiana, U. cornuta, and U. rubrotincta formed distinct clades by clustering with some of the specimens used in this study. Some of the Usnea species did not form any distinct clades: Group I included 25 species of Usnea namely U. acanthella, U. acromelana, U. antarctica, U. aurantiacoatra, U. baileyi, U. diffracta, U. filipendula, U. flavocardia, U. florida, U. fulvoreagens, U. hirta, U. glabrescens, U. lambii, U. patagonica, U. pectinata, U. perpusilla, U. silesiaca, U. sphacelata, U. subantarctica, U. subfloridana, U. subrubicunda, U. trachycarpa, U. trichodeoides, U. ushuaiensis, and U. wasmuthii; Group II included five species of Usnea namely U. brasiliensis, U. erinacea, U. rubricornuta, U. rubicunda, and U. steineri; Group III included three species of Usnea namely U. articulata, U. crocata, and U. subcornuta; Group IV included two species of Usnea namely U. fragilescens and U. glabrata; U. ceratina and U. perhispidella formed two separated mono-taxa clades; N (red colour): Two clades formed by Neuropogon species in this phylogenetic tree.
Figure 4.9 Lineage-through-time (LTT) plot (left) and phylogenetic tree analysed based on the estimated threshold (right) investigated for mycobiont of Usnea specimens (including ITS rDNA of 45 species of Usnea from GenBank and 367 specimens of Usnea generated in this study) using General Mixed-Yule Coalescent approach in the single threshold mode. The sudden rise in branching rate was considered as the transition from interspecific to intraspecific region (red line) and the delimited lineages were shown using different colours in phylogenetic tree.
Figure 4.10 Lineage-through-time (LTT) plot (left) and phylogenetic tree analysed based on the estimated thresholds (right) investigated for mycobiont of Usnea specimens (including ITS rDNA of 45 species of Usnea from GenBank and 367 specimens of Usnea generated in this study) using General Mixed-Yule Coalescent approach in the multiple thresholds mode. The sudden rise in branching rate was considered as the transition from interspecific to intraspecific region (red lines) and the delimited lineages were shown using different colours in phylogenetic tree.