IJSEM Papers in Press. Published November 18, 2011 as doi:10.1099/ijs.0.037028-0
1
Pseudonocardia xishanensis sp. nov., a novel endophytic actinomycete
2
isolated from the roots of Artemisia annua L.
3 4
Guo-Zhen Zhao1†, Jie Li 2†, Wen-Yong Zhu1, Da-Qiao Wei3, Jin-Li Zhang1,
5
Li-Hua Xu1, Wen-Jun Li1
6 7
1
8
Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of
9
Microbiology, Yunnan University, Kunming, 650091, PR China.
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education and
10
2
11
Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China
12
Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
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Guangzhou, 510301, PR China.
14
3
15
Kunming University of Science and Technology 650500, Kunming, 650224, PR China.
Key Laboratory of Marine Bio-resources Sustainable Utilization, CAS, RNAM Center for
Faculty of Environmental Science and Engineering & Life science and Technology of
16 17
Author for correspondence: Wen-Jun Li
18
Tel & Fax: +86 871 5033335
19
E-Mail:
[email protected];
[email protected]
20 21
†
These authors contributed equally to this work.
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Running title: Pseudonocardia xishanensis sp. nov.
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Category: New Taxa – Actinobacteria
26 27
The 16S rRNA gene sequence of strain YIM 63638T has been deposited in GenBank
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under the accession number FJ817397.
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A Gram-positive, non-motile, endophytic actinomycete, designated strain YIM
30
63638T, was isolated from the surface-sterilized roots of Artemisia annua L., and
31
its taxonomic position was investigated by a polyphasic approach. The isolate
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grew optimally with 1–3 % (w/v) NaCl, at pH 6.0-7.0 and at 20–37°C.
33
Comparative analysis of the 16S rRNA gene sequence showed that the isolate
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belongs to the genus Pseudonocardia, showing the highest level of sequence
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similarity with respect to Pseudonocardia oroxyli D10T (98.9 %), Pseudonocardia
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ailaonensis YIM 45505T (98.3 %) and Pseudonocardia halophobica DSM 43089T
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(98.0 %). The phylogenetic distances from other described species with validly
38
published names within the genus Pseudonocardia were greater than 2.3 %.
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Strain YIM 63638T had a genomic DNA G+C content of 72.1 mol% and
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MK-8(H4) as the predominant respiratory quinone. The major fatty acids were
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iso-C16:0 (36.32 %) and 10-methyl-C16:0 (19.78 %). On the basis of its phenotypic
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properties and phylogenetic distinctiveness, strain YIM 63638T represents a
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novel species of the genus Pseudonocardia, for which the name Pseudonocardia
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xishanensis sp. nov. is proposed. The type strain is YIM 63638T (=JCM
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17906T=KCTC 29005T).
46 47
The genus Pseudonocardia (family Pseudonocardiaceae) was described by Henssen
48
(1957), and since then the description of the genus has been revised repeatedly
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(Warwick et al., 1994; McVeigh et al., 1994; Reichert et al., 1998; Huang et al., 2002;
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Park et al., 2008). Members of the genus Pseudonocardia display vegetative and
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aerial mycelium with spore chains produced by acropetal budding or fragmentation.
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Pseudonocardia strains have a type IV cell wall, the major menaquinone is MK-8 (H4)
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or MK-9 and the DNA G+C content is 68–79 mol%. Members of the genus
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Pseudonocardia have been widely reported and recovered from several ecosystems,
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such as soil samples (including those polluted by industrial chemicals) and plant
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samples (including stems, leaves, root nodules, tree-bark compost and Chinese
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traditional medicine plants).
58
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During the course of a study on endophytic actinomycete diversity and bioactive
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metabolites isolated from a Chinese traditional medicine plant, a large number of
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novel bacterial strains were isolated. In this study, we characterized one of these
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isolates, strain YIM 63638T. Phenotypic, chemotaxonomic and phylogenetic analyses
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established the affiliation of isolate YIM 63638T to the genus Pseudonocardia. The
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data obtained also suggest that this isolate represents a novel species of the genus
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Pseudonocardia.
66 67
Healthy root samples of Artemisia annua L., a Chinese traditional medicine plant,
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were collected from Xishan Mountain in Kunming, Yunnan province, south-west
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China, and used as sources for isolation of endophytic actinomycetes. The plant
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samples were air-dried at room temperature after thorough washing under tap water
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and were then surface-sterilized according to the procedure described by Li et al.
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(2008). The isolation method involved processing of 1 g of plant sample in a
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commercial blender, followed by grinding with a mortar and pestle, adding 9 ml
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sterile water, and serial dilution of 1 ml tissue suspension to 10-2, before plating on
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agar plates. The isolation medium was humic acid-vitamin (HV) agar (Hayakawa &
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Nonomura, 1987) supplemented with nalidixic acid (25 mg l-1) and nystatin (50 mg l-1)
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[Please indicate the temperature and duration of incubation on HV agar plates to
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isolate strain YIM 63638]. A pure culture was obtained by repeated streaking on 1/2
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strength International Streptomyces Project medium 2 (ISP 2, Shirling & Gottlieb,
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1966). The purified strain YIM 63638T was routinely cultivated on ISP 2 medium at
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28°C and stored as glycerol suspensions (20%, v/v) at -80 °C.
82 83
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA
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gene were performed as described by Li et al. (2007). The 16S rRNA gene sequence
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of strain YIM 63638T was compared against a database of cultured species via
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BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in order to retrieve the most
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similar sequences of bacteria with validly published names. Multiple alignments were
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performed with the CLUSTAL_X software package (Thompson et al., 1997) and the
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Kimura two-parameter model (Kimura, 1983) was used to calculate evolutionary
90
distances. The phylogenetic trees were constructed by the neighbour-joining (Saitou
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&
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maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981)
93
tree-making algorithms by using the software packages MEGA version 4.0 (Tamura
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et al., 2007) and PHYML (Guindon & Gascuel, 2003). The topologies of the
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phylogenetic trees were evaluated by using the bootstrap resampling method of
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Felsenstein (1985) with 1000 replicates. The 16S rRNA gene sequence of Kutzneria
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kofuensis NRRL B-24061T (AF114801) was used as an outgroup. DNA–DNA
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relatedness was studied using the fluorometric micro-well method (Ezaki et al., 1989;
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Christensen et al., 2000; He et al., 2005), and the hybridizations were performed with
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Nei,
1987),
minimum-evolution
(Rzhetsky
&
Nei,
1992,
1993),
six replicates.
101 102
The almost-complete 16S rRNA gene sequence (1521 nt) of strain YIM 63638T was
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aligned manually with the corresponding partial 16S rRNA gene sequences of the type
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strains of representative members of the genus Pseudonocardia retrieved from
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GenBank/EMBL/DDBJ. Phylogenetic analyses based on 16S rRNA gene sequences
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showed that strain YIM 63638T fell into a distinct subclade with the type strains of
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Pseudonocardia oroxyli D10T, Pseudonocardia ailaonensis YIM 45505T and
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Pseudonocardia halophobica DSM 43089T, an association which was supported by
109
all of the tree-making algorithms employed (Fig. 1., Fig. S2, Fig. S3 and Fig. S4).
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Strain YIM 63638T shared highest 16S rRNA gene sequence similarity with P. oroxyli
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D10T (98.9 %), which corresponds to 15 nt differences at 1413 locations, and lower
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similarities with P. ailaonensis YIM 45505T (98.3 %) and P. halophobica DSM
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43089T (98.0 %). Lower levels of sequence similarity (≤ 97.7 %) were found to other
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type strains of the genus Pseudonocardia. DNA–DNA hybridization tests were
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carried out between strain YIM 63638T and the phylogenetically most closely related
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type strains. The DNA–DNA relatedness values between YIM 63638T and P. oroxyli
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D10T (31.0 ± 1.7 %), P. halophobica DSM 43089T (27.8 ± 0.9 %) and P. ailaonensis
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YIM 45505T (25.5 ± 1.5 %) were all significantly lower than 70%, the threshold value
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for the delineation of genomic species (Stackebrandt & Goebel, 1994), thus
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suggesting that strain YIM 63638T should be considered as a representative of a
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separate species.
122 123
Morphological, cultural, physiological and biochemical characteristics of strain YIM
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63638T were studied by following the guidelines of the International Streptomyces
125
Project (ISP; Shirling & Gottlieb, 1966). Gram staining was carried out by using the
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standard Gram reaction, and cell motility was assessed by the development of
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turbidity throughout a tube containing semisolid medium (Leifson, 1960). The
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morphological characteristics were observed by light microscopy (BH2; Olympus)
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and scanning electron microscopy (Philips XL30; ESEM-TMP) using cultures grown
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on ISP 2 medium at 28°C for 7-14 days. Cultural characteristics were recorded on ISP
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media, Czapek’s agar, potato-glucose agar and nutrient agar, which were prepared as
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described by Dong & Cai (2001). Colours were determined by using the colour chips
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from the ISCC–NBS colour charts (standard samples, no. 2106) (Kelly, 1964).
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Phenotypic characteristics of strain YIM 63638T and the phylogenetically closest
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neighbours were examined using several standard methods. Temperature range
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(4–55°C), pH (4.0–10.0) and NaCl (0–20 %, w/v) tolerance for growth were
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determined as described by Zhao et al. (2011b). Catalase activity, oxidase and
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gelatinase activities, starch hydrolysis, nitrate reduction and urease were assessed as
139
described by Smibert & Krieg (1994). Media and procedures used for the
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determination of other physiological characteristics, carbon source utilization and acid
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production from carbohydrates were those described by Gordon et al. (1974).
142 143
Cells of strain YIM 63638T were Gram-positive, strictly aerobic, non-motile,
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oxidase-negative and catalase-positive. Growth was observed at 10–42°C (optimum
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20–37°C), pH 5.0–9.0 (optimum pH 6.0–7.0) and 0–7 % (w/v) NaCl (optimum
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1-3 %). The strain grew well in the basal mineral salts medium (Pridham & Gottlieb,
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1948) with L-arabinose, D-cellobiose, D-fructose, D-galactose, glucose, glycerol,
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myo-inositol, lactose, maltose, D-mannitol, D-mannose, L-rhamnose, D-sorbitol,
149
sucrose and D-xylose as the sole carbon and energy source. Additional growth factors
150
(vitamins or amino acids) were not required. Growth was not observed when dulcitol,
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D-raffinose, D-ribose or sodium acetate was supplied as the sole carbon and energy
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source. Acid was produced from D-fructose, D-galactose, glucose, lactose, maltose,
153
D-mannose
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L-hydroxyproline, hypoxanthine, L-lysine, L-phenylalanine, L-serine, L-tyrosine,
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L-valine and xanthine were used as sole nitrogen sources. Strain YIM 63638T
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developed well on most media tested, including ISP 2, ISP 3, ISP 4, ISP 5, Czapek’s
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agar, nutrient agar and potato-glucose agar media. The strain produced white aerial
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mycelium on all the media tested. The colour of the substrate mycelium was
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yellow–brown on ISP 5, Czapek’s agar and potato-glucose agar media and
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orange–yellow on ISP 2, ISP 3, ISP 4 and nutrient agar media. A diffusible pigment
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(brown-yellow) was observed on potato-glucose agar. Morphological observation of
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14 day-old cultures of strain YIM 63638T revealed that both aerial and vegetative
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hyphae were abundant, well developed and fragmented into rod-shaped elements
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(Supplementary Fig. S1). Activities of urease, catalase and hydrolysis of starch and
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Tweens 20, 40 and 80 were positive. Tests for H2S production, milk coagulation, milk
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peptonization, nitrate reduction, oxidase and hydrolysis of cellulose and gelatin were
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negative. Phenotypic characteristics that differentiate strain YIM 63638T from closely
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related type strains are given in Table 1.
and
L-rhamnose.
L-alanine,
L-arginine,
L-asparagine,
glycine,
169 170
The isomer of diaminopimelic acid and the sugars in whole-cell hydrolysates were
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determined according to the procedures described by Hasegawa et al. (1983),
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Lechevalier & Lechevalier (1970) and Tang et al. (2009). Polar lipids were extracted,
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examined by two-dimensional TLC and identified using previously described
174
procedures (Minnikin et al., 1979). Menaquinones were isolated according to Collins
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et al., (1977) and separated by HPLC (Tamaoka et al., 1983). Mycolic acids were
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extracted and analyzed by one-dimensional TLC as described by Minnikin et al.
177
(1980). Cellular fatty acids were extracted, methylated and analysed by using the
178
Sherlock Microbial Identification System (MIDI) according to the manufacturer’s
179
instructions. The fatty acid methyl esters were analysed by using the Microbial
180
Identification software package (Sherlock Version 4.0; MIDI database: TSBA40).The
181
G+C content of the genomic DNA was determined by using the HPLC method
182
(Mesbah et al., 1989) with E. coli JM-109 as the reference strain.
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Whole cell-wall hydrolysates of strain YIM 63638T were rich in meso-diaminopimelic
184
acid, arabinose, galactose, glucose and mannose (cell-wall chemotype IV; Lechevalier
185
& Lechevalier, 1970). MK-8(H4) was detected as the major respiratory quinone of
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strain YIM 63638T, which is characteristic of species of the genus Pseudonocardia.
187
Mycolic acids were absent. Strain YIM 63638T possessed iso-C16:0 (36.32 %) and
188
10-methyl-C16:0 (19.78 %) as the major fatty acids. In addition, iso-C15:0
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2-OH/C16:1ω7c (7.76 %), C16:0 (7.65 %), iso-C16:1 H (7.51 %) and C17:1ω6c (7.16 %)
190
were produced in moderate amounts. The major polar lipids of strain YIM 63638T
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comprised
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phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylcholine,
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phosphatidylinositol, an unknown glycolipid and two unknown polar lipids. The DNA
194
G+C content of strain YIM 63638T was 72.1 mol%.
diphosphatidylglycerol,
phosphatidylglycerol,
195 196
The results of the phylogenetic analysis and morphological and chemotaxonomic
197
investigations support the affiliation of strain YIM 63638T to the genus
198
Pseudonocardia. Differences in biochemical characteristics, 16S rRNA gene
199
sequences, isolation source and DNA G + C content can be used to distinguish strain
200
YIM 63638T from phylogenetically related taxa. Therefore, on the basis of these data,
201
strain YIM 63638T merits recognition as the type strain of a novel species in the genus
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Pseudonocardia, for which the name Pseudonocardia xishanensis sp. nov. is
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proposed.
204 205
Description of Pseudonocardia xishanensis sp. nov.
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Pseudonocardia xishanensis (xi.shan.en′sis. N.L. fem. adj. xishanensis of or
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belonging to Xishan Mountain, the source of the plant Artemisia annua L. from which
208
the type strain was isolated).
209 210
Aerobic, non-motile, Gram-positive actinomycete that forms extensively branched
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substrate and aerial mycelia. Mycelia fragment into rod-shaped elements. Forms white
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aerial mycelium and yellow–brown or orange-yellow substrate mycelium on media
213
tested. A diffusible pigment (brown-yellow) is observed on potato-glucose agar. The
214
temperature range for growth is 10-42 °C, with optimal growth occurring at 20-37 °C.
215
The pH range for growth is 5.0-9.0 (optimum, pH 6.0–7.0). The NaCl concentration
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range for growth is 0-7 % (optimum 1-3 %). Positive for hydrolysis of starch, catalase
217
and urease, but negative for milk coagulation and milk peptonization, nitrate
218
reduction, oxidase, gelatin liquefaction, cellulose hydrolysis and H2S production.
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Tweens 20, 40 and 80 are hydrolyzed. Utilizes L-arabinose, D-cellobiose, D-fructose,
220
D-galactose,
221
D-mannose, L-rhamnose, D-sorbitol, sucrose and D-xylose as sole carbon and energy
222
sources. Dulcitol, D-raffinose, D-ribose and sodium acetate are not utilized. L-alanine,
223
L-arginine, L-asparagine, glycine, L-hydroxyproline, hypoxanthine, L-lysine,
224
L-phenylalanine, L-serine, L-tyrosine, L-valine and xanthine can be used as sole
225
nitrogen sources. Acid is produced from D-fructose, D-galactose, glucose, lactose,
226
maltose, D-mannose and L-rhamnose. The cell wall contains meso-diaminopimelic
227
acid (meso-DAP). The whole-cell sugar pattern consists of arabinose, galactose,
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glucose and mannose. MK-8(H4) is the predominant menaquinone. Mycolic acids are
229
absent. The polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol,
230
phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylcholine,
231
phosphatidylinositol, an unknown glycolipid and two unknown polar lipids. The
232
major fatty acids are iso-C16:0 (36.32 %) and 10-methyl-C16:0 (19.78 %). The G+C
233
content of the genomic DNA is 72.1 mol%.
glucose,
glycerol,
myo-inositol,
lactose,
maltose,
D-mannitol,
234 235
The type strain, YIM 63638T (=JCM 17906T=KCTC 29005T) was isolated from
236
surface-sterilized roots of Artemisia annua L. collected from Xishan Mountain,
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Kunming City, Yunnan province, south-west China.
238 239
Acknowledgements
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This research was supported by the National Natural Science Foundation of China
241
(No. U0932601).
242
243
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Warwick, S., Bowen, T., McVeigh, H. P. & Embley, T. M. (1994). A phylogenetic
346
analysis of the family Pseudonocardiaceae and the genera Actinokineospora and
347
Saccharothrix with 16S rRNA sequences and a proposal to combine the genera
348
Amycolata and Pseudonocardia in an emended genus Pseudonocardia. Int J Syst
349
Bacteriol 44, 293–299.
350
Zhao, G. Z., Zhu, W.Y., Li, J., Xie, Q., Xu, L. H. & Li, W. J. (2011a).
351
Pseudonocardia serianimatus sp. nov., a novel actinomycete isolated from the
352
surface-sterilized leaves of Artemisia annua L. Antonie van Leeuwenhoek. 100(4):
353
521-528.
354
Zhao, G. Z., Li, J., Zhu, W.Y., Li, X. P., Tian, S.Z., Zhao, L.X., Xu, L. H. & Li,
355
W. J. (2011b). Pseudonocardia bannaensis sp. nov., a novel actinomycete isolated
356
from the surface-sterilized roots of Artemisia annua L. Antonie van Leeuwenhoek 100
357
(1), 35–42.
358 359 360 361
362
Table 1. Different physiological characteristics of strain P. xishanensis YIM 63638T
363
and closely related type strains.
364
Strains: 1, P. xishanensis YIM 63638T; 2, P. oroxyli D10T; 3, P. halophobica DSM
365
43089T; 4, P. ailaonensis YIM 45505T. Data were obtained in this study.
366
Characteristic
1
2
3
4
dulcitol
-
+
+
+
myo-Inositol
+
+
-
+
D-mannitol
+
+
-
+
L-raffinose
-
+
-
+
D-ribose
-
+
+
+
D-sorbitol
+
+
-
+
sucrose
+
+
-
+
D-xylose
+
+
+
-
+ +
-
+
L-hydroxyproline
+
+
+ w w
+
glycine
+
-
+
+
+
-
-
-
Gelatin liquefaction
-
-
+
+
Nitrate reduction
-
-
-
+
-
-
w
42 ºC
w w
-
w
-
pH 5.0
w
-
w
-
7% NaCl
w
w
+
-
-
-
w
-
Utilization as sole carbon source:
Utilization as sole nitrogen source: L-arginine
Hydrolysis of: Urease Starch
Growth at/on: 10 ºC
10% NaCl
367 368
Note: +, positive or present; w, weakly positive; -, negative or absent.
+ -
P. xishanensis YIM 63638T (FJ817397)
83
P. oroxyli D10T (DQ343154)
89 93
P. ailaonensis YIM 45505T (DQ344632) P. halophobica IMSNU 21327T (AJ252827) P. tetrahydrofuranoxydans k1 T (AJ249200) 99
100
P. dioxanivorans CB1190T (AY340622) P. benzenivorans B5T (AJ556156)
79 54
P. sulfidoxydans DSM 44248T (Y08537) P. hydrocarbonoxydans IMSNU 22140T (AJ252826)
369
0.005
370
Fig. 1. Neighbour-joining tree of Pseudonocardia xishanensis YIM 63638T sp. nov.
371
and related species based on 16S rRNA gene sequences. Bar, 0.005 substitutions per
372
nucleotide position.
373
Supplementary materials
374 375 376
Table S1. Fatty acid profiles (%) of strains P. xishanensis YIM 63638T and the
377
closely related type strains. Fatty acid C14: 0 C15: 0 C16: 0 C17: 0 C17: 1ω6c iso-C14: 0 iso-C15: 0 iso-C16: 0 iso-C17: 0 iso-C16: 1 H iso-C16: 02-OH C16:1ω7c/C15:0iso2-OH anteiso-C15: 0 anteiso-C17: 0 C16: 0 10-methyl C17: 0 10-methyl
1 1.27 1.11 7.65 0.44 7.16 3.37 2.42 36.32 0.56 7.51 7.76 0.39 0.96 19.78 0.97
2 1.66 4.34 2.50 2.86 49.49 0.83 12.99 11.50 10.50 -
3 0.57 1.96 6.28 1.03 14.50 0.68 4.56 34.78 2.55 5.91 3.57 3.43 1.32 6.64 7.36 1.17
4 1.81 8.38 5.22 1.63 7.76 35.51 4.68 6.91 10.75 1.56 8.99 -
378
Strains: 1, P. xishanensis YIM 63638T; 2, P. oroxyli D10T; 3, P. halophobica DSM
379
43089T; 4, P. ailaonensis YIM 45505T.
380
Note: -, negative or absent.
381 382 383
384 385 386
Fig.S1. Scanning electron micrographs of strain YIM 63638T grown on ISP 2
387
medium for 2 weeks at 28 °C. Bars, 5 or 10 μm.
388 389
P. kunmingensis YIM 63158T (FJ817377)
100 *
P. sichuanensis KLBMP 1115T (HM153789) P. zijingensis DSM 44774T (AF325725) P. adelaidensis DSM 45352T (FJ805427) P. serianimatus YIM 63233T (FJ817379)
95 * 64 * 99 *
P. petroleophila IMSNU 22072T (AJ252828) P. aurantiaca CCTCC AA97002T (AF056707)
* 51 *
P. xinjiangensis CCTCC AA97020T (EU722520) P. alaniniphila YIM 16303T (EU722519) P. yunnanensis IFO 15681T (D85472)
100 *
P. mongoliensis MN08-A0270T (AB521671) P. saturnea IMSNU 20052T (AJ252829) 100 *
P. artemisiae YIM 63587T (GU227146) P. bannaensis YIM 63101T (FJ817375)
P. chloroethenivorans SL-1T (AF454510) P. asaccharolytica DSM 44247T (Y08536) P. babensis VN05A0561T (AB514449) P. spinosispora LM 141T (AJ249206)
65 *
P. spinosa JCM 3136T (AB547126) P. acaciae GMKU095T (EU921261) P. eucalypti EUM 374T (FJ805426)
61 56
100 79 64 88 95 *
88 71 71
99 98
83 89 93 *
P. compacta IMSNU 20111T (AJ252825) P. autotrophica IMSNU 20050T (AJ252824) P. kongjuensis LM 157 T (AJ252833) P. ammonioxydans AS 4.1877T (AY500143)
P. endophytica YIM 56035T (DQ887489) P. parietis 04-St-002T (FM863703) P. tropica YIM 61452T (GQ906587) P. nitrificans IFAM 379T (X55609) P. antarctica DVS 5a1T (AJ576010) P. carboxydivorans SWP-2006T (EF114314) P. alni IMSNU 20049T (AJ252823) Pseudonocardia xishanensis YIM 63638T (FJ817397) P. oroxyli D10T (DQ343154) P. ailaonensis YIM 45505T (DQ344632)
P. halophobica IMSNU 21327T (AJ252827)
*
P. tetrahydrofuranoxydans k1T (AJ249200) P. dioxanivorans CB1190T (AY340622) 100
99 *
79 54 96 * 100
P. benzenivorans B5T (AJ556156) P. sulfidoxydans DSM 44248T (Y08537)
P. hydrocarbonoxydans IMSNU 22140T (AJ252826) P. thermophila IMSNU 20112T (AJ252830) P. khuvsgulensis MN08-A0297T (AB521672) P. rhizophila YIM 67013T (GU322368) K. kofuensis NRRL B-24061T (AF114801)
0.005
390
391
Fig. S2. Phylogenetic relationships of strain YIM 63638T and other closely related
392
Pseudonocardia species based on 16S rRNA gene sequences. The branching pattern
393
was generated by the neighbour-joining method. Asterisks indicate branches
394
recovered
395
maximum-parsimony and maximum-likelihood. Bootstrap values (expressed as
396
percentages of 1000 replications) of above 50% are shown at branch points. Bar,
397
0.005 substitutions per nucleotide position.
398 399 400 401 402
with
four
methods:
neighbour-joining,
minimum-evolution,
