Russian Journalof Nematology, 2011, 19 (1), 21 - 29
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Identification and intraspecific variability of Steinernema feltiae strains from Cemoro Lawang village in Eastern Java, Indonesia Ternesgen Addis!, Mulawarman Mulawarman2, Lieven Waeyenberge3, Maurice Moens 3,4, Nicole Viaene3 and Ralf-Udo Ehlers 5 IDepartment of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium, e-mail:
[email protected] 2Department of Plant Protection, Faculty of Agriculture, Sriwijaya University, Jl.Simpang Indralaya KM 32, 30662, Palembang, Indonesia, 3Plant-Crop Protection, Institute for Agricultural & Fisheries Research, Burg. Van Gansberghelaan 96 bus 2, Merelbeke, Belgium, 4Laboratory for Agrozoology, Ghent University, Coupure links 653, 9000 Ghent, Belgium, SDepartment of Biotechnology & Biological Control, Institute for Phytopathology, Christian-Albrechts-University,Hermann-Rodewald-Str. 9,24118 Kiel, Germany Accepted for publication 28 October 2010
Summary. Four strains of Steinernema feltiae from Eastem Java, Indonesia were characterised based on morphometric, morphological and molecular data. In addition, their virulence against last instar Tenebl'io molitor and heat toleranee was tested. Infective juvenile have a mean body length ranging from 749 to 792 Ilm. The maximum sequence difference among the four strains was 7 bp (8.8%) in the ITS and 2 bp (0.3%) in D2D3 regions of the rDNA. All the strains are not reproductively isolated and can reproduce with European strain S.feltiae Owiplant. The lowest LC so was observed for strain SCM (373) and the highest for S. fettiae strain Owiplant (458) infective juveniles. All the four strams showed relatively better mean heat toleranee when compared with S. feltiae Owiplant both in adapted and non-adapted heat tolerance experiments. Key words: Cross hybridisation, D2D3, ITS, morphometrics, phylogeny, taxonomy, Tenebl'io molitol', virulen e.
Entomopathogenic nematodes (EPN) together with their associated enteric bacteria Xenol'habdus and Photorhabdu, respectively, are lethal pathogens of everal economically important insect p sts (Grewal et al., 2005). Many surveys have been conducted all over the world in search of species of EPN (e.g. Hominick, 2002; Grewal et al., 2005). EPN exhibit great variation in their infectivity and survival. Collecting indigenous nematodes can possibly provide isolates more suitable for the contral of local pests because of their adaptation to local c1imatic conditions and population regulators (Stock et al., 2008). The huge natural genetic diversity in EPN populations around the world can also be exploited for breeding programmes to improve beneficia1 traits of EPN for biological control. However, one of the fITst and most important needs for use in biocontrol or for dome tication of EPN IS their accurate
identification. New methods using sequence analysis of Intemal Transcribed Spaeer (lTS) and the D2D3 expansion regions of rDNA have proven to be useful for identification and molecular analysis of nematodes (Spiridonov et al., 2004; Nadler et al., 2006). The objectives of this study were to identify intraspecific variability of the strains using molecular, morphometric, morphological and cross hybridisation methods and to compare their virulence and heat toleranee with a European isolate of S. feltiae.
MATERIAL AND METHODS Nematodes. Steinernema feltiae strains SCM, SNGD, SNC and Ssp60 were obtained from Cemoro Lawang village, Eastem Java, Indonesia at 2,329 m above sea level using the Gallel'ia baiting technique (Bedding & Akhurst, 1975). Steinernema feltiae strain Owiplant from Poland, kindly provided by
21
Temesgen Addis et al.
Dr.Marek Tomalak, Poznan, Poland, was used to compare virulence and heat tolerance with the newly identified Steinernema feltiae strains. Morphological and morphometric characteri sation. Ten Galleria mellonella larvae were exposed in a Petri dish (100 x 15 mm) lined with two moistened filter papers to 2000 infective juveniles (IJ) (Nguyen, 2007). First and second generation adult nematodes were obtained by dissecting infected insects in Ringer's solution 2-4 days and 5-7 days, respectively, aftel' the host had died. lnfective juveniles were obtained when they emerged from the cadavers aftel' 7-10 days using a White trap (White, 1927). Specimens of different stages were killed and fixed in TAF (Courtney et al., 1955). Nematodes fixed in TAF were processed to glycerin by the Seinhorst method (Seinhorst, 1959). Morphometric analysis of the nematode specimens were made for 20 individual males, 20 females and 20 IJ using light microscopy and image analysing software (CeW'D, Olympus soft imaging solutions GmbH, Germany). The species were identified by comparing data with species descriptions and keys by Nguyen (2007) and Nguyen et al. (2007). For morphometric data analysis measurements of IJ and first generation males were used. Molecular characterisation and phylogeny. DNA was extracted from three pooled live IJ using the method reported by Spiridonov et al. (2004). The lTS regions ofthe ribosomal DNA (rDNA) was amplified by PCR. The PCR reaction mixture contained 5 111 of lQx PCR reaction buffer, 2 mM MgCh, 200 IlM of each dNTP, 1 IlM forward and reverse primer, 0.4 111 (2 UIlrl) Taq Polymerase (Invitrogen, Merelbeke, Belgium), 5 111 crude DNA extract and ddH 20 up to a volume of 50 ~d. The primers TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') as forward and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') as reverse primer (Joyce et al., 1994) were used. The D2D3 region was amplified by PCR using the same reaction mixture but with the primers being replaced by D2A (5' ACA AGT ACC GTG AGG GAA AGT TG-3 ') as forward and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') as reverse primers (De Ley et al., 1999). The PCR product was cloned in a pGEM®-T Vector and JM 109 I-{igh Efficiency Competent Cells according to the manufacturer's instructions (Part number TM042, Promega, USA). P1asmid DNA was purified based on the method described in the Pure Yield Plasmid Miniprep System (Prornega). Quantification of both the PCR-product and tbe plasmid DNA was done by putting 2 111 of tbe
22
product on a UV-spectropbotometer (Nanodrop 100). An approximate 20 111 purified plasmid DNA with PCR-insert (100 ng Ilr1) was sent to a sequencing service (Macrogen, Seoul, South-Korea) for sequencing. Sequence data of the four strains SCM, SNC, SNGD and Ssp60 were edited using BioEdit (BIOEDIT version 7.0.9, lnvitrogen). The sequences of the four strains together with those available in the GenBank were aligned using the default parameters of Clusta1 X (Thompson et al., 1997). Phylogenetic trees and pairwise comparisons were obtained by Maximum Parsimony (MP) using PAUP, 4.0b8 (Swofford, 2002). All data were assumed to be unordered, all characters were treated as equally weighted, gaps as missing data. Maximum parsimony was performed with a heuristic search with 1000 replicates (simple addition sequence, stepwise addition, tree-bisection reconnection (TBR) branch swapping). For the lTS sequences, Caenorhabditis elegans (X03680) was used as out-group taxon (Nguyen et al., 2001). For D2D3, Panagrellus redivivus (AF331910) was used as out-group taxon (Stock et al., 2001). Branch support was estimated by bootstrap analysis: One thousand replicates for MP and 10,000 replicates for neighbour joining (NJ) (Nguyen et al., 2001, Nguyen, 2007). Trees were displayed with TreeView1.6.1 (Page, 1996). Cross hybridisation. Twenty males of one nematode strain were cultured on Nutrient Lipid Agar (Wouts, 1981) plates with 20 pre-mature females of another strain and vice versa. In each of tbe combinations, the symbiotic bacteria were taken from the female partner. Ten G. mellonella last instal' larvae were inoculated with 100 IJ per G. mellonella in a Petri dish (100 x 15 mm) with moist filter paper and kept at 25°C in dark room. At 12-24 h aftel' inoculation, the infected G. mellonella was sterilised using 95% ethanol. The cadaver was then opened with a sterile needie and a drop of haemolymph was streaked on to NBTA agar (Akhurst, 1980). Aftel' sub-culturing and confirming the absence of contaminants, a single colony was transferred in to BSA liquid medium (Ehiers et al., 1998). The bacterium was incubated for 1-3 days at 25°C in the dark. Virulence and heat toleranee tests. Vinllenc was assessed by determination of the Iethal concentrations (LC) of four strains from lndonesia and S. feltiae OwipIant. Doses of 0, 50, 100, 200, 400 and 800 IJ in 1 mI water were used to inoculate 40 meaIwonns of Tenebrio molitor L. (Co1eoptera: Tenebrionictae) in Petri dishes filled with moist sand
and incuba Larval mo fOffi1Ula (A, calculated To adap h prior to b left to rec exposed to ranging fro water was ~ one hour experiment batcbe of bottom of t PtlOO thin I n matodes r ever an active nema a water trap
I
Morpho characterisa ranging frarr head to excl ariance sh morphometri the character ob ened in t :s 0.05; Tab\! different frol R, and AB length (P :S ignificantly the other stra In fir t ge the strains ra Ilm) to the b o tati tica: four strain~ width. GL an (P:S 0.05; Ta Molecula Maximum pa sam con en S C Ssp60 form a mono] value of 99 é M. S Gl ( Fl \050) Sleinemema of 100°'0 ani criteria (Fig. In the MF S ,and S
Steinernemafeltiae variability in Eastem Java
opNA ) a
:ea) ~.ins nng ~he
lose tlle al., ;ons sing vere
ated Iata.
t~~;
lTS was For used anch
One
sfor .001, with one jpid
ature ~h
of
:aken
last G. 110ist 12-24 I was then p of agar ming r was ~t al., lYS at ~r
lence
lethal mesia ,200, culate ptera: t sand
and incubated at 2SoC according to Peters (200S). Larval mortality was corrected using Abbott's formuia (Abbott, 1925). LC so and LC go values were calculated using Probit analysis (Finney, 1971). Ta adapt IJ to heat, they were kept at 3SoC for 3 h prior to heat stress treatment. Afterwards, IJ were left to recover for I h at 2SoC and were then exposed to five different temperature gradients ranging from 37°C to 41°C for 2 h. Two mi tap water was added on the five cover-slide chambers atle hour ahead of introducing 200 IJ. This experiment was repeated three times with different batches of nematodes. The temperature on the bottom of the chambers was recorded by Platinum PtlOO thin layer sensors. After the heat treatment the nematodes were left in the dark at 2SoC ovemight to recover and counting was done after separating active nematodes from the dormant nematodes using a water trap (Ehiers et al., 200S).
RESULTS Morphological and morphometric characterisation. IJ had a mean body length ranging from 749-792 Ilm and S3-S4 distances from head to excretory pore (TabIe 1). The analysis of variance showed no significant difference in morphometric data among the four strains in most of the characters. However, significant difference was observed in body length between Ssp60 and SNC (P :s O.OS; Table I). In addition, Ssp60 is significantly different from the other strains in body diameter, NR, and ABD and significantly shorter in hyaline length (P ::s O.OS; Table 1). Strain SNGD has a significantly longer tail length when compared with the other strains (P::S O.OS; Table 1). In first generation males the mean body length of the strains ranges from the lowest for SNGD (1,081 Ilm) to the highest for SCM (1,243 Ilm) (TabIe 1). No statistically significant differences between the four strains were recorded in ES, T, SL, spicule width, GL and gubemaculum width , D% and SW% (P:S O.OS; Table 1). Molecular characterisation and phylogeny. Maximum parsimony and NJ criterion produced the same consensus tree in which the strains SCM, SNC, Ssp60 and S. feltiae strain SN (AF1210S0) farm a monophyletic group supported by a bootstrap value of 99 and 78%, respectively (Fig. la). SNC, SCM, SNGD, Ssp60 and S. feltiae strain SN (AF1210S0) were distinct from the other Steinernerna species tested, with a bootstrap value of 100% and 94%, respectively, in MP and NJ criteria (Fig. 1). In the MP consensus tree, the four strains SNC, SCM, and SNGD were grouped together with S.
feltiae strain SN with a bootstrapping support of 6S%. However, the strain Ssp60 is out of this grouping. Steinernerna rnonticolurn and S. ashiuense are distinct from the other Steinernerna species and the four strains tested with a bootstrap support of 90% (Fig. 1b). Pairwise comparison of the sequences of the ITS regions of the rDNA indicates that the four strains are closely related with each other and with S. feltiae strain SN. Between S. feltiae strain SN and SNGD a 99.2% similarity ratio and a 99.9% with the remaining three strains was found (TabIe 2). The similarity ratio between the tested species ranged between 81.1 % (s. feltiae strain SN and S. ashiuense) to 96.1 % (s. feltiae strain SN and S. litorale) (Tabie 2). Similarly, comparison of the sequences of D2D3 region of rDNA of S. feltiae strain Bodega Bay and the four strains showed 99.8% to 100% similarity ratio (Tabie 2). The similarity ratio of the remaining Steinernerna species with S. feltiae strain Bodega Bay was from 93.2% (between S. feltiae and S. rnonticolurn) to 99.6% (between S. feltiae and S. silvaticurn). There is no difference in the D2D3 regions of rDNA of S. feltiae strain Bodega Bay with SNC and SNGD and only one bp with SCM and Ssp60 (TabIe 2). Cross hybridisation. In the cross breeding tests, involving males of each of the four strains and females of S. feltiae strain Owiplant, copulation started after a few minutes of introduction on agar plates. Similarly, in reverse crosses between females of each strain with males of S. feltiae strain Owiplant copulation was observed. In both crosses between males and females and reverse crosses of each of the strains with S. feltiae Owiplant offspring were produced starting from the fourth day after introduction of males and virgin females. Further observation on the offspring obtained from the crosses and reverse crosses was continued every day and the offspring were found to be fertile and produced another generation of nematodes. Virulence and heat toleranee tests. The lowest LC so was observed with strain SCM (373 IJ) and the highest LC so with S. feltiae strain Owiplant (4S8 IJ) followed by SNC (408 IJ) per 40 mealworms (Tabie 3). Based on LC so and LC go values there was no significant difference (P ::s O.OS) in virulence between the strains studied when compared with S. feltiae strain Owiplant. The four strains showed relatively better heat tolerance than S. feltiae strain Owiplant, both in the adapted and non-adapted heat tolerance experiments. Moreover, all the strains including S. feltiae Owiplant showed an increased tolerance after
23
IV .j::>.
Table 1. Cornparison of rnorphometric characters of infective juveniles and fust gen~ration rnales of Steinernema feltiae strains SCM, SNC, SNGD , Ssp60 and those provided by the bibliography. All rneasurernents are in Jlrn (rnean ± SD (range)). Means followed by tbe same letter in rows are not significantly different from each other; according to Tukey's HSD test, P ? 0.05. *After Nguyen et al. (2006). ** After Carnpos-Herrera et al. (2006). - Data not available.
N L
S. feltiae (Rioia)"" -
SCM
SNC
20
S. feltiae (SN)" 20
First generation males SNGD SspGO
20
20
20
20
792±58 b (684-900)
879± 49 (766-928)
783 ± 75.3 (660-914)
1243 ± 96 b (1109-1363)
1081 ± 101 (892-1253) a
32 ± 4.0 b (26-40) 7 i 0.5 b (6-8) 10 ± 0.5 a (10-11) 6 ± 0.7 b (4-8) 25 ± 3.0 a 119-30) 57 ± 4.9 b (52-73) 91 ±4.4a (81-100) 120 ± 6.5 a (105-133)
29± 2.1 a (24-33) 6±1 a (4-7) II ±0.7 b (10-13) 5 ± 0.9 a (4-7) 28±l.lb (25-29) 54 ± 3.6 ab (49-61) 101 ± 16.4 b (87-135) 133 ± 16.5 e (115-165)
30± 1.9 (27-34) 6.4 ± 0.3 (5.8-6.8) 10 ± 0.5 (9.4 -11) 4.8 ± 0.2 4.5-5. I) 29 ± 1.9 (26-32) 63 ± 2.3 (58-67) 113 ±5.1 (108-117) 136 ± 3.5 (130-143)
30 ± 3.3 (20_37' 6.6 ± 0.5 (5.9-6.7) 11 ± 0.7 (10-13)
26± 2.9 (20-30) 64 ± 10.6 (50-89) 93 ± 6.5 (80-105) 118 ± 8.3 (102-138)
12 ± 1.7 b (9-14) 8 ± 0.6 b (7-9) 46± 5.6 b (35-54) 0.6 ± 0.1 a (0.5- 0.8) 106 ± 13.4 b (82-135) 81±6.7b (65-88) 115 ± 5.3 b (104- 122) 154 ± 5.2 a (144-162)
1142 ± 122 (801-1293) ab lO±l.la (7-12) 8 ± 0.9 ab (6-10) 41 ±6.3 b (28-52) 0.6±0.la (0.4-0.8) IJ8 ± 17.1 b (82-146) 77 ± 9.5 ab (57-93) L13 ± 8.1 b (98- 128) 153 ± 12.3 a (l30- 168)
12± 0.6 b (11-14) 7 ±0.6 a (6-8) 35 ± 3.5 a (29-41 ) 0.7±0.1 b (0.6-0.9) 88 ± 6.8 a (80-101) 79±6.8b (62-92) 113 ± 6.1 b (103-125) 148 ± 7.2 a (134-160)
1169± 101 ab (L0341405) 1I ± 0.8 a (9-13) 8 ±0.6 b (7-10) 43 ± 8.9 b (32-68) 0.6±0.1 a (0.4-0.7) 112± 11.9 b (84-130) 71 ±9.5 a (55-93) 105±4.7a (97- 1(2) 146 ± 6.2 a (138-165)
76 ±4.6 b (67-85) 46 ±4.8 b (36-59, 12 ± 1.8 ab (10-19)
72 ± 5.6 a (61-80) 39 ± 3.0 a (34-46) 14 ± 2.2 e (10-17)
86± 2.6 (81-89) 38 ± 3.2 (32-43) 18 ±0.8 (16-19)
72 ± 5.5 (61-80)
27 ± 4.4 a (23-38)
28 ±3.S a (22-35)
31 ± 3.1 a (26-39)
-
-
-
-
47±2.1ab (44-50) 69± 3.6 a (61-74) 14± 1.3 a (12-16) 47±3.0a (41-51) a 7± 0.8 a (6-9) 52 ± 4.3 a (42-58) 302 ± 43.5 b (222-358) 136 ± 38.9 a (123-161) 67 ± 6.2 a (52-74)
49±3.7b (43-57) 69±3.7a (61-77) 17±10.3a (48-10) 48±4.7a (37-56) 7± 0.9 a (5-8) 51 ±9.1 a (34-69) 275 ± 39.2 ab (362-194) 142 ± 11.5 a (126-162) 70 ± 6.9 ab (53-82)
lnfective Juveniles SspGO SNGD
Character SCM
SNC
20
20
20
759 ± 58 ab (647-832)
749 ± 46a (665-807)
789 ± 43 ab (693-868)
32±3.1 b (27-38) 6 ± 0.4 ab (6-7) 11 ±0.8 ab (9-12) 6 ± 1.2 a (4-8) 24 ± 1.8 a (20-26) 53 ±4.7 a (44-61) 92 ± 8.5 a (76 -105) 123 ± 12 ab (98-142)
33 ± 5.1 b (27-43) 6 ±0.5 a (5-7) II ±0.7ab (10-13) 6 ± 0.9 b (4-8) 23 ± 2.9 a (18-28) 53 ± 2.4 a (49-57) 94±6.7ab (84-112) 132 ± 8.0 be (119-151) 69 ±4.9 a (59-76) 44±4.7b (35-53) II ± 1.8 a (8-16)
I I
a b c c' Body Diameter
EP NR ES
70±3.4a (62-76) 44 ±4.6 b (32-53) 13±2.1 he (9-16)
T H ABD
-
-
Spicule Length (SL) spicule width
-
-
Gubemaculum length (GL)
-
-
-
43 ± 3.3 a (37-49) 76±6.6 a (64-83) 63 ±5.0 b (52-73)
D% (EPIES X 100) E% (EPrr X 100) SW% (SU ABD X 100)
41 ±3.2a (33-46) 78 ±4.0 a (73-86) 63 ±4.2 b (57-74)
47 ±4.9 b (40-60) 75±7.Ja (65- 98) 61 ±4.4 b (50-69)
54 ± 7.7 (40-69) 88 ± 10.4 (75-109) s. feltiae (Rioja)"
-
-
Gubemaculum width
-
41 ±6.1 a (31-47) 75 ± 5.6 a (66-87) 55 ± 3.4 a (49-62)
-
-
46 ± 1.4 (44- 50) 74±4 (67-81) 44±4 (37-51)
783 ± 75.3 (660-914) 30 ± 3.3 (20-37' 6.6 ± 0.5 (5.9-6.7) II ±0.7 (10-\3)
GS% (GUSL X 100)
44±2.9a (40-51) 68 ± 4.2 a (60-76) 13 ± 1.5 a (10-15) 49 ± 4.3 a (39-55) 7 ± 0.9 a (5-9) 54 ± 4.2 a (43-62) 258 ± 27.5 a (190-294) 153 ± 12.8 a (132-173) 72 ± 5.4 ab (60-80)
I
I
I
I
~
I
~
I
•
I
~
I
I
_
I
(l)
::: ;J> 0 0
S. feltiae (SN)" 20
S feltiae (Rioia)""
1612 ± 88 (14141815) 11.5
1220 ± 176.4 (820- L648)
9.5 41.3
~
~
12 ± 1.7 (8-17) i 9.3 ± 1.5 . (7-12.6) 51 ± 7.9 (40-70)
0.8 140 ± 10 (121-162) 115±3.4 (110-126)
107 ± 20.9 (73-141) 86 ±9.9 (61-97)
170 ± 3.4 (l64-180i
138 ± 12.4 (100-152)
28±4.1 a (18-34) -
39 ± 1.2 (37-43)
28 ± 3.5 (21-33)
47±2.7ab (41-53) 67 ± 3.2 a (58-71) 13 ± 1.7 a (10-16) 50 ± 2.8 a (45-55) 7 ± 0.7 a (5-8) 49 ± 6.8 a (38-63) 26l ±55.9ab (178-382) 143± Il.7a (110-170) 75±4.lb (66-83)
48 ± 1.7 (43-53) 66± 1.5 (62-68)
l I L
'J> (IQ
v;'
34 ± 3 (28-39) 68 ±4.6 (58-80) -
52± 1.9 (48-56)
47 ± 3.7 (41-53)
68 ±3.1 (64-72)
62 ± 6.7 (46-70)
140± 10 (130-150) 80±3 (70-90)
205 ± 80 (169-235) 70 ± 5.8 (60-83)
Table 2. Pair-wise distancl:S oflTS and 0203 regions ofrONA bClween species infeltiac-group and slrains SCM, SNC, SNGO and Ssp60. Numbers below diagonal are lotal character difTerences and above diagonal mcan ChaTlll:ter difTcrcnccs (adjusted lor missing data).
_ _ _ _ _ _ _ _ _ _oll...:so,,"-.Clo:
~ 3
(l)
L
I
I
Table 2. Pair-wise distances oflTS and 0203 regions ofrONA between species infeltiae-group and strains SCM, SNC, SNGO and SspGO. Numbers below diagonal are tota! character differences and above diagona! mean character differences (adjusted for missing data).
Species lTS region
N Ul
I
2
3
4
5
6
7
8
9
10
11
12
13
I
S. monticolum
-
007581
0.15632
0.16480
0.15907
0.18443
0.18579
018194
0.15580
0.17720
0.18939
0.14716
0.51043
2
S. ashiuense
68
-
0.19344
0.19451
019523
0.20055
0.20192
0.20083
0.18940
0.19337
0.19909
0.18544
0.50785
3
S. kraussei
141
171
-
0.06492
0.04145
0.09159
0.09159
0.09045
0.07423
0.08668
008516
0.07193
0.49556
4
S. texanum
147
170
62
-
0.06540
0.10614
0.10486
0.10486
0.08691
0.10128
0.10070
0.08765
0.48686
5
S. oregonense
143
172
40
62
-
0.08132
0.08132
008015
0.06562
0.07761
0.06964
0.06112
0.49121
6
SNC
135
146
73
83
64
-
000248
0.00248
0.00124
0.00878
0.05089
0.04835
0.47690
7
SCM
136
147
73
82
64
2
-
0.00248
0.00124
0.00878
0.05089
0.04835
0.47441
8
Ssp60
133
146
72
82
63
2
2
-
0.00124
0.00754
0.04952
0.04707
0.47625
9
S. feltiae
141
168
72
83
63
I
I
I
-
0.00753
0.04952
0.03858
0.47331
10
SNGD
129
140
69
79
61
7
7
6
6
-
0.04127
0.04198
0.47601
II
S. weiseri
125
131
62
72
50
37
37
36
36
30
-
0.03320
0.49309
12
S. litorale
132
163
72
83
58
38
38
37
37
33
24
-
0.47944
453
502
463
475
382
380
381
461
377
357
478
-
13
C. e1egans
465
D2D3 region 1
SNC
-
0.00000
0.00162
0.00162
0.02435
0.00370
0.00000
0.01020
0.01533
0.00832
0.04708
0.05236
0.27094
2
SNGD
0
-
0.00162
0.00162
0.02435
0.00370
0.00000
0.01020
0.01533
0.00832
0.04708
0.05236
0.27094
3
SCM
1
I
-
0.00325
0.02597
0.00556
000170
0.01190
0.01704
0.00998
0.04870
0.05410
0.26929
4
Ssp60
I
I
2
-
0.002273
0.00185
0.00170
0.00850
0.01363
0.00666
0.04545
0.05061
0.26929
5
S. texanum
15
15
16
14
-
0.02186
0.02791
0.3023
003613
0.02864
0.06840
0.06036
0.28669
6
S. silvaticum
2
2
3
I
12
-
0.00364
001093
001642
0.00546
0.04372
0.04736
0.28885
7
S. feltiae
0
0
I
I
24
2
-
0.01269
00'1736
0.00577
0.06799
0.04812
0.29466
8
S. oregonense
6
6
7
5
26
6
11
-
0.01042
0.01384
0.06551
0.04695
0.29316
9
S. kraussei
9
9
10
8
31
9
15
9
-
0.01852
0.07054
0.05300
0.29569
10
S. weiseri
5
5
6
4
25
3
5
12
16
-
0.06326
0.04408
0.29558
11
S. monticolum
29
29
30
28
58
24
55
53
57
52
-
0.03019
0.29688
12
S. ashiuense
30
30
31
29
51
26
41
40
45
38
24
-
0.30565
13
P. redivivus
165
165
164
164
252
158
254
253
254
261
247
265
-
S:2
'"S· ~
~."
~
~
'
