Molecular characterization and phylogenetics of entomopathogenic nematodes (nematoda: stienernematidae) from Asia

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1 Molecular characterization and phylogenetics of entomopathogenic nematodes (nematoda: stienernematidae) from Asia Item Type text; Electronic Thesis Authors Cho, Hyun-Bae Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 14/06/ :43:26 Link to Item

2 MOLECULAR CHARACTERIZATION AND PHYLOGENETICS OF ENTOMOPATHOGENIC NEMATODES (NEMATODA: STEINERNEMATIDAE) FROM ASIA By HYUN-BAE CHO A Thesis Submitted to The Honors College In Partial Fulfillment of the Bachelor s degree With Honors in Molecular and Cellular Biology THE UNIVERSITY OF ARIZONA May 2009 Approved by: Dr. S. Patricia Stock Department of Entomology 1

3 STATEMENT BY AUTHOR I hereby grant to the University of Arizona Library the nonexclusive worldwide right to reproduce and distribute my thesis and abstract (herein, the licensed materials ), in whole or in part, in any and all media of distribution and in any format in existence now or developed in the future. I represent and warrant to the University of Arizona that the licensed materials are my original work that I am the sole owner of all rights in and to the licensed materials, and that none of the licensed materials infringe or violate the rights of others. I further represent that I have obtained all necessary rights to permit the University of Arizona Library to reproduce and distribute any nonpublic third party software necessary to access, display, run, or print my thesis. I acknowledge that University of Arizona Library may elect not to distribute my thesis in digital format if, in its reasonable judgment, it believes all such rights have not been secured. SIGNED: 2

4 INDEX Page Abstract 4 Introduction 4 Materials and methods 1.Nematode propagation 10 2.Harvesting of infective juvenile nematodes(ijs) 11 3.Recovery of adult nematode stages 12 4.Extraction of Nucleic Acids 12 5.Polymerase Chain Reactions 14 6.Gel electrophoresis 16 7.Preparation of PCR products for sequencing 17 8.Sequence submission, diting and alignments 18 9.Phylogenetic analysis 18 Results 1.Molecular identification of studied isolates 19 2.Phylogenetic analysis 32 3.Morphological analysis 36 Discussion 40 Acknowledgements 41 References cited 42 3

5 ABSTRACT In this study I molecularly characterized 13 entomopathogenic nematode isolates from various countries in Asia, including South Korea, India, Turkey, and Syria. Two nuclear genes, the large subunit of (LSU or28s) and the Internal Transcribed Spacer (ITS) region of ribosomal DNA were considered for this purpose. Approximately, 1Kb and 800bp were amplified for 28S and ITS genes, respectively. Obtained sequences were compared with an existing library in PI Stock s laboratory and also with sequences available in GenBank. Evolutionary relationships between the unknown isolates and known and described species were also assessed. Currently molecular systematics is an important tool for diagnosis and identification of many plants and animals. Moreover, molecular phylogenetic analysis can also be considered to map and interpret the evolution of many traits including biological, ecological behavioral, biogeography, among others. INTRODUCTION Nematodes are one of the most abundant and ubiquitous animals on earth (Bongers and Ferris, 1999, Lambshead, 1999). They have been able to adapt to nearly every type of environment from fresh water to marine, polar region to the tropics, and low to high elevations. They can be free-living in terrestrial, freshwater and marine environments, feeding on bacteria fungi and other microorganisms. One of the most important groups are plant parasites, they cause billions in dollars of damage of crops worldwide. Likewise, vertebrates (of humans and other vertebrates) are considered very important in causing and transmission of many diseases in developing countries (Poinar, 1979). Other groups are associated with insects, mites and mollusks of potential importance in agriculture, forestry or health (Poinar, 1983; Petersen, 1985; Gaugler and Kaya, 1990; Bedding, 1993; Wilson et al., 1993, 1994; Wilson and Gaugler, 2000; Grewal et al., 2003). 4

6 Nematode association with invertebrates span from casual (i.e. phoretic, commensal) to obligate parasitism and pathogenesis. According to Poinar (1983), invertebrate parasitism arose in four major groups of nematodes. He stated that one of the most primitive groups, the Rhabditida, gave rise to members of the Oxyurida (c. 420 million years ago) as well as to the Steinernematidae and Heterorhabditidae (375 million years ago) (Figure 1). Poinar (1993) also speculated that morphological and life history similarities between these two groups were the result of convergent evolution. Based on similarities of the buccal capsule and male tail morphology, Poinar (1993) suggested that steinernematids have evolved from a terrestrial proto- Rhabditonema ancestor, while heterorhabditids arose from a Pellioiditis-like ancestor in a sandy marine environment. The notion that heterorhabditids and steinernematids do not share an exclusive common ancestor has been proposed by other studies based on cladistic interpretation of morphological traits (Sudhaus, 1993) and of molecular data (Adams et al., 1998), and a combination of both approaches (Liu et al., 1997). Poinar (1983) also suggested that plant parasitic tylenchids gave rise to the Allantonematidae (300 million years ago) and Sphaerulariidae (223 million years ago) and that the insect-parasitic Entaphephelenchidae probably arose from an aphelenchoid ancestor approximately 300 million years ago (Figure 1). The fourth group of invertebrate parasites for which Poinar (1983) suggested an evolutionary hypothesis was the Mermithida, where he suggested predaceous dorylaimids as their closest ancestors (185 million years ago) (Figure 1). According to Blaxter invertebrate parasitism arose independently at least four times in the evolution of Nematoda (Figure 2). One of the most studied groups of invertebrate parasites are the insect pathogenic or entomopathogenic nematodes (EPN). These nematodes belong to two families: Heterorhabditidae and Steinernematidae (Hominick, 2002; Hominick et al., 1996). The Nematoda evolutionary framework based on 18S rdna sequence data (Blaxter et al., 1998) supported Poinar s (1993) shows two independent origins of steinernematids and heterorhabditids. According to this analysis, Steinernema is most closely related to the Panagrolaimidae (mostly free-living nematodes, with some genera considered as insect associates) and Strongyloides (vertebrate parasites). These three groups (Steinernematidae, Panagrolaimidae and 5

7 Strongyloididae) are members of a larger clade that comprises plant parasitic, fungal-feeding and bacterivorous taxa of the order Tylenchida, Aphelenchida and Cephalobida (Figure 2). Figure 1. Schematic representation of the evolution of invertebrate parasitism in Nematoda according to Poinar (1983) (From Stock and Hunt, 2005) The same tree also showed Heterorhabditis as being most closely related to the Strongylida (vertebrate parasites), both clades sharing the rhabditoid Pellioditis (Rhabditida) as their most recent common ancestor (Figure 2). Blaxter et al. s tree placed mermithids, another group of insect parasites, as being most closely related to the free-living mononchids, and as a member of a larger clade that included the vertebrate-parasitic trichocephalids and the plantparasitic trichocephalids and the plant-parasitic dorylaimids (Figure2). These results are consistent with Poinar s hypothesis of a predatory dorylaimid as the closest relative to mermithids. Entomopathogenic nematodes are obligate and lethal parasites of insects. These nematodes are associated with gram-negative symbiotic bacteria (Enterobacteriaceae: γ-proteobacteria) in the genera Photorhabdus (for Heterorhabditidae) and Xenorhabdus (for Steinernematidae). These nematodes have an interesting life cycle. The third stage dauer juvenile (IJ) occurs free in the soil and its role is to seek out and infect an insect larva. Steinernema gains entry to the insect larva through natural openings (mouth, anus and spiracles). In addition to these modes of 6

8 entry, Heterorhabditis juveniles also gain entry by abrading the intersegmental membranes of the insect the IJ release cells of a symbiont bacterium that it carries in its intestine. Figure 2. Schematic representation of the evolution of invertebrate parasitism in Nematoda (From Stock and Hunt, 2005) AOP=algivore-omnivore-predator; B=bacterivore; EP=entomopathogen; F=fungivore; IP=invertebrate parasite; PP=plant parasite; VP=vertebrate parasite,*=used or with potential as biocontrol agents.(from Stock and Hunt, 2005) 7

9 The insect haemolymph provides rich medium for the bacterial cells and these begin to grow, release toxins and exoenzymes and kill the insect. The insect dies rapidly, usually within h. The nematodes resume development, moult to the J4 stage and reach adulthood within 2 (S. carpocapsae) or 3 (H. bacteriophora) days when cultured in vivo in larvae of the greater wax moth Galleria mellonella at 23 C (Wang & Bedding, 1996). Nematode reproduction continues over two to three generations until the nutrient status of the cadaver deteriorates whereupon adult development is suppressed and IJ accumulates. These non-feeding infective stages emerge into the soil where they may survive for several months in the absence of a suitable host. Both EPN families have a worldwide distribution; the only continent where they have not been found is Antarctica (Griffin et al., 1990). But at the same time, factors such as soil texture, moisture content, temperature, and host availability are thought to be important in determining distribution of entomopathogenic nematodes (Hominick and Briscoe, 1990; Hara et al., 1991; Hominick et al., 1996; Simoes and Rosa, 1996; Steiner, 1996; Stock et al., 1999) The number of newly discovered nematode species and isolates with biocontrol potential has significantly increased over the past decade (Stock and Hunt, 2005; Hunt and Nguyen, 2008). Accurate identification diagnosis of these taxa requires the consideration of appropriate taxonomic tools. To meet these expectations nematode systematists have incorporated new technologies into their traditional morphological approaches including several molecular techniques (Stock and Hunt, 2005). Several molecular techniques have been considered in nematode systematic. Many of these approaches have provided interesting and important insights into biodiversity and evolution, particularly for parasitic nematodes such as Steinernematidae and Heterorhabditidae (Akhurst, 1987; Reid and Hominick, 1992; Gardner et al., 1994; Liu and Berry, 1995; Liu et al., 1997; Reid et al., 1997; Adams et al., 1998; Nguyen et al., 2001; Stock et al., 2001). At present, nuclear rdna genes are considered useful markers for delimitation of nematodes at different taxonomic levels (e.g. Curran and Driver, 1994; Blaxter et al., 1998; Nadler and Hudspeth, 1998, 2000). Three regions of nuclear rdna that are widely used are 18S or Small Subunit (SSU) gene of rdna, internal transcribed spacer (ITS) region and 5.8 gene of 8

10 rdna, and 28S or Larger Subunit (LSU) of rdna. For example, the 18S or SSU gene of rdna has demonstrated to be too conserved in resolving relationships among Heterorhabditis (Liu et al., 1997) or Steinernema (Stock et al., 2001). The ITS region has also been used in EPN systematics. This variable region has revealed numerous diagnostic markers, especially for Heterorhabditidae. The 28S or LSU of rdna gene has also been used to assess phylogenetic relationships among Steinernema spp. (Stock et al., 2001). This region is also considered to be so far the effective and reliable approach for delimitation of terminal taxa in Steinernema as well as diagnostic purposes (Stock et al., 2001; Stock and Hunt, 2005). 9

11 MATERIALS AND METHODS 1. Nematode propagation Nematodes considered in this study originated from soil samples collected in four countries of Asia: Turkey, Syria, India and South Korea (See Table 1). Infective juvenile nematodes were established in the laboratory by rearing them in vivo using the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae) according to procedures described by Kaya and Stock (1997). Infection chambers were built following procedures described by Kaya and Stock (1997). Briefly, 10 cm Petri dishes previously sterilized with 95% ethanol were used up-side-down and two discs of filter paper Whatman # 1) were placed on the bottom dish. For each culture, approximately 1ml of nematode suspension at a concentration of 1,000-3,000 nematodes/ml was applied onto the filter paper. Ten G. mellonella larvae were added to the dish. The dishes were closed with the upper lid and placed in plastic bags to prevent loss of moisture, and incubated at 25 C for 2 to 3 days, and until mortality of the G. mellonella larvae occurred. Infection chambers were placed in an incubator at 25 C. Insect mortality was checked within in hours after initial infection. Dead insects are usually recognized by change of their coloration. steinernematids have a typical dark cream to light brown color, whereas heterorhabditids have a brick-red to burgundy tone (Figure 3). After 3-4 days, dead insects were placed in modified White traps (water trap) (Kaya and Stock, 1997) (Figure 4). 10

12 Table 1. List of EPN isolates considered in this study Country of Origin Isolate code EPN Family Turkey CK-6, Tur-S3, K4, 60, KRK3, R03, R04,B02 Steinernematidae India S01 H02 Steinernematidae, Heterorhabditidae Syria 5C, D3 Steinernematidae South Korea 75-2 Steinernematidae Figure 3. G. mellonella being infected by isolate CK-6 (S. feltiae) Figure 4. White trap is set up after nematode harvesting is done. 2. Harvesting of infective juvenile nematodes (IJs) Nematodes were harvested from White traps by removing the inner dish that contained the cadavers and pouring the suspension of IJs into a beaker. The dish containing the cadavers was placed back into the larger Petri dish and more water was added to the larger dish. Once the IJs in the beaker settle to the bottom, the supernatant water can be decanted and new water was added to the beaker. This process was repeated three times. 11

13 3. Recovery of adult nematode stages The timeframe for the dissections varied depending on the different nematode species and/or isolates. For steinernematids, dissections were done in 2-3 days after infection to get the first generation adults. Dissections were performed in Petri dishes with M9 buffer solution (Brenner, 1974) and under a dissecting microscope at a magnification of 20-50X (Figure 5 A). Galleria cadaver were cut using a dissecting needles, by pressing down the body of the insect with one needle, and using another needle to cut off the insect s head. The degraded insect contents (no organs can be recognized in this phase as the nematode bacterial symbionts have degraded all organs in the insect hosts) were then examined to recovered nematodes inside. Adults male and females were pulled out and placed in a watch glass filled with M9 buffer (Figure 5 B). Approximately nematodes per species/isolated were collected. Three rinses with M9 buffer were done, to make sure a clean nematode suspension was done. Nematodes were then transferred to a 1.7 ml micro-centrifuge tube with TE buffer ph 8.0 for subsequent DNA extraction. The tubes were placed in a micro-centrifuge at 13,000 rpm for 5 min. to allow nematodes to settle in the bottom. This process was repeated 3 times. Nematodes were then stored in -20C freezer in 500ul of TE buffer for future DNA extraction. 4. Extraction of Nucleic Acids A standard phenol-chloroform method was considered for DNA extraction of EPN isolates following procedures described by Stock et al., Micro-centrifuge tubes (1.5 ml) containing bulk of IJs or adult stages were thaw from the freezer and 15µl of 30% SDS and 20 µl of proteinase K were added into the tube containing 500 µl of TE buffer ph 8.0. Nematodes were grinded inside tubes with small rod-shaped grinder and later incubated at 50 C in a water bath until all tissues were fully digested. Once digestion of tissues was completed, 10 µl of RNase were added to each sample to remove RNA. The sample was then vortexed and incubated at 12

14 37 C for one hour. The micro-centrifuge tube was then spun at 4,000 rpm for 2 min, and the supernatant was transferred to a new 1.7 ml micro-centrifuge tube. Figure 5A. Adult nematodes within dead tissue of Galleria (magnification10x) Figure 5B. Collection of nematodes in a watch glass filled with M9 buffer Following this step 500 µl of phenol (molecular grade) was added to each sample, which was then vortexed briefly and spun at 13,000 rpm for 5 min. The supernatant was transferred to the new tubes and repeat this step 3 more times. The upper supernatant was then transferred to a new tubes and 500 µl of chloroform/ isoamyl alcohol (24:1) was added to each sample. The tubes were vortexed briefly and spun at 13,000 rpm for 5 min. The upper interface was removed carefully and transferred to a new micro-centrifuge tube. Na acetate (1M) in a proportion of 10 µl per 100 µl of sample was added to help precipitation of salts. Absolute (100%) ethanol was the added to the tube placed in freezer overnight. The following day, samples were spun for 10 min at 13,000 rpm and the supernatant was discarded. The tubes were placed in desiccators to dry and samples were then resuspended in 25 µl of TE ph 8.0. DNA concentration was read using a Shimatzu spectrophotometer, and the DNA concentration was then adjusted to 100 ng/ µl for use in PCR reaction. 13

15 5. Polymerase Chain Reactions Polymerase chain reaction (PCR) is a very widely used method for nucleic acid amplification. To prepare the PCR reactions we considered Red Taq PCR mix (Sigma R RXN), the total volume of a PCR mix in a tube is adjusted to 25µl. PCR conditioning, e.g., annealing temperature and MgCl2 concentration, were adjusted empirically to optimize reaction specificity for individual species. Based on the DNA concentration data from spectrophotometer, the volume of DNA sample in PCR mix may vary. The different amount of DNA samples between each tube was compensated with MQ H2O volume. For example, if 1.0µl of DNA sample was used due to the high concentration of DNA, then 9.5µl of MQ H20 was used to keep the total volume of 25µl. However, if sample had lower concentration of DNA (< 100 ng/µl, then 8.5µl of MQ H2Owas used. 12.5µl of PCR Red Taq. Primers were usually used at a total volume of and 1.0µl/each (forward and reverse) for each PCR reaction tube. Steinernematidae Primers: For steinernematid nematodes, portion of the nuclear large-subunit (LSU) ribosomal DNA (approximately 1Kbp) that includes the D2 and D3 domains was considered (Stock et al., 2001) (Figure 6). The forward and reverse primers for amplifying 28S rdna were: 391 Forward PCR primer (5 -AGCGGAGGAAAAGAAACTAA, positions 3,745-3,764 in Caenorhabditis elegans GenBank X03680) 501 Reverse PCR primer (5 -TCGGAAGGAACCAGCTACTA, positions 4,681-4,700) designed by Tomas et al. (1997). 502 Internal forward primer (5 -CAAGTACCGTGAGGGAAAGTTGC) (Stock et al 2001) 503 Internal reverse primer (5 -CCTTGGTCCGTGTTTCAAGACG) (Stock et al 2001) PCR conditions were the following: denaturation at 94 C for 3 minutes, followed by 33 cycles of 94 C for 30 seconds, 52 C for 30 seconds, and 72 C for 1 minute, followed by a postamplification extension at 72 C for 7 minutes. 14

16 Heterorhabditidae Primers: For Heterorhabditid nematodes, a portion of the internal transcribed spacer (ITS) region of the tandem repeat unit of rdna was considered (Adams et al., 1998). Two sets of primers were considered for amplification the ITS gene, depending on the nematode isolate. Primer set I: 93 Forward PCR primer (5 -TTGAACCGGGTAAAAGTCG, positions 2,635-2,653) (Nadler & Holdberg et al. 2000) 94 Reverse PCR primer (5 -TTAGTTTCTTTTCCTCCGCT, positions 3,745-3,764) (Nadler & Holdberg et al. 2000) Primer set II AB28- Forward PCR primer (5 -ATATGCTTAAGTTCAGCGGGT) (Curran et al. 1994) TW81- Reverse PCR primer (5 -GTTTCCGTAGGTGAACCTGC) (Curran et al.1994) KN58- Internal forward primer (5 -GTATGTTTCCTTGAAGGTC) (Nguyen et al. 2001) KNRV- Internal reverse primer (5 -CACGCTCATACAACTGCTC) (Nguyen et al. 2001) PCR conditions were the following: denaturation at 94 C for 3 minutes, followed by 33 cycles of 94 C for 30 seconds, 60 C for 30 seconds, and 72 C for 1 minute 15 seconds, followed by a postamplification extension at 72 C for 5 minutes. A negative control for checking any contamination, and a positive control (from a sample that has already worked out for the same reaction condition) were always considered. 15

17 S ITS 5.8 S ITS 2 28 S AB28 KNRV KN58 TW = ITS variable region = Highly conserved region = Moderately conserved region Figure 6. This figure shows relative region where each primer would bind to amplify DNA. Purple and blue color primers are for ITS region, and green color primers are for 28S rdna region. 6. Gel electrophoresis A 1% agarose gel was considered for electrophoresis of PCR products. To make a 1% gel the following reagents were considered: 0.45g of agarose, mixed with 45ml of 1X TAE (Trisacetate-EDTA) buffer. Ingredients were poured into a beaker and heated until boiling. After cooling down, the agarose solution was poured into the molding set up, and allowed to solidify. The gel was then loaded with a ladder (1Kb), the PCR samples and the negative and positive controls as well. SYBR green I dye was used instead of Et-Br, because it allows gels to be reused. The SYBR green is added to the samples and not the gel. Finally, the gel was run at 80V for 20 min, and then visualize on gel box using SYBR Green filter (Figure 7). 16

18 Figure 7. 1% gel showing positive PCR products Lane 1: ladder 1kb Lane 2: Isolate S01 with 391/501 primers Lane 3: Isolate S01 with 391/501 primers Lane 4: Isolate H02 with 93/94 primers Lane 5: Isolate H02 with 93/94 primers Lane 6: Positive control Lane 7: Negative control This example shows successful PCR reactions (bright bands) and their approximate size. These PCR products are sent to the sequencing facility after ExoSAP-IT treatment (see section below). 7. Preparation of PCR products for sequencing When the gel electrophoresis showed that the samples had the desired concentration of DNA, PCR products were prepared for sequencing. For this purpose we considered an enzymatic digestion method with ExoSAP-IT (manufactured by USB Corp..). This product is used to cleanup PCR products ranging in size from less than 100bp up to over 20kb. PCR products are not lost when using this enzyme because the product is not subjected to a purification method. ExoSAP- IT enzymes degrade all the reagents in PCR product such as primers, and RedTaq and leave the DNA sample clean. For PCR cleaning we added 5µl of PCR product and 2µl of ExoSAP-IT to a total of 7.0µl. The mixture was placed in a thermal cycler and incubated at 37 C for 15 min, to 17

19 activate the enzymes. And then a second heating phase at 80 C for 15 min was followed to kill enzymes. PCR products were then submitted to DNA sequencing. 8. Sequence Submission and Alignment procedures Samples were submitted to the Arizona Research Labs Sequencing Facility on campus. For this purpose, primers used for the PCR reactions were diluted to 3µM. Five microliters of primers were prepared for each samples. Samples and primers were then submitted to the DNA sequencing facility. Successfully sequenced samples were carefully edited and ambiguity resolution was performed with the aid of Seq Edit and SeqMan software v.7.2 (Lasergene, DNAStar Corp.) Sequence segments corresponding to the PCR amplification primers were removed prior to multiple sequence alignment. Sequences were aligned initially using CLUSTAL X version 1.53b (Thompson et al., 1997), and the resulting output was adjusted manually to improve homology statement considering McClade software (Madison and Madison, 2000). 9. Phylogenetic analysis Only Steinernematidae sequences were considered to assess phylogentic relationships. For this purpose, obtained LSU sequences for all Steinernema isolates were subjected to analysis, using heuristic searches (simple stepwise addition, TBR branch-swapping, MULPARS) and 1,000 replicates. An existing library (P. Stock s laboratory, University of Arizona) of more that 50 Steinernema spp. and available sequences deposited in GenBank were considered for inferring phylogenetic relationships of studied isolates. Phylogenetic analyses (maximum parsimony analysis) of sequence data were made using PAUP* v 4.0b (Swofford, 2001) following criteria described by Stock et al. (2001) and Nadler et al. (2006). Caenorhabditis elegans was considered in both phylogenetic analyses as the outgroup taxon according to criteria described by Nadler et al. (2006). 18

20 RESULTS 1. Molecular Identification of Studied Isolates Sequences were compared with an existing database of EPN species in Stock s lab. Below is the result of CLUSTAL X alignment. Stars (*) indicate similar base pairs across taxa. CLUSTAL X (1.83) multiple sequence alignment S.affine TAGGATTTCT-CTAGTAACTGCGAGTGAAACGGAA S.intermedium GGGGAGGAAAGAAACTAACTAGGATTTCT-CTAGTAACTGCGAGTGAAACGGAA S.feltiae60 --TAGCGGAGGAAAAGAAACTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeB02 --TAGCGGAGGAAAAGAAACTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeK4 ---AGCGGAGGAAAAGAAACTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeTurS GGAGGAAAAGAAACTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeCK6 -TTAGYGGAGGAAAAGAAACTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiae GGNGGAAAGGAAACTAACTTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiae5C CTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeKrK TAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeR CTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.feltiaeR GAGTGAAACGGAA S.puntauvense TAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.kraussei -----TCGAGGAAA-GAAACTAACT-AGGATT--C-TTAGTAACTGCGAGTGAAACGGAA S.oregonense -TTACCGGAGGAAAAGAAACTAACTTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.kushidai --TAACCGGAGAAAAGAAACTAACT-AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.diaprepesi AAAAGAAACTAACTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.puertoricense --TAAGCGGAGGAAAAGAAACTAACTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA Stein TAGCGGAGGAAAAGAAACTAACTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.cubanum TAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.longicaudum GGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.glaseri GGAGGAAAAGAAACTAACTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.arenarium GGAGAAAAGAAACTAACCTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.hermaphroditum TTCC-TTAGTAACTGCGAGTGAAACGGAA S.scarabaei GGATTTYC-TTAGTAACTGCGAGTGAAACGGAA S.khoisanae TCC-TTAGTAACGGCGAGTGAAACGGAA S.karii AGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.rarum --TTACCGGAGGAAAAGAAACTAACTAGGATTTCC-TTAGTAACTGCGAGTGAAACGGAA S.costaricense -----CCGGGGAAAAAGAAACTAAMWMGGAYTTCC-TTAGTAACTGCGAGTGACAAGGAA S.bicornutum CTAGGATTCC-TTAGTAACTGCGAGTGAAACGGGA S.ceratophorum TAGATTCTC-TTAGTAACTGCGAGTGAAAAGGGA S.abassi GGATTTCC-TTAGTAACTGCGAGTGAAAAGGAA S.abbasi-S01-Iran AACTAACTAGGATTTCC-TTAGTAACTGCGAGTGAAAAGGAA S.riobrave --TAANCSGAGGAAAAGAAACTAACTAGGATTGCC-TTAGTAACTGCGAGTGAAACGGCA S.websteri TAGGATTTCC-TTAGTAACTGCGAGTGAAAAGGAA S.anatoliense ---TTAGGAGGGAAAAGAAACTAACTAGGATTTCCCTTAGTAACTGCGAGTGAAAAGGAA S.monticolum CTAGGATTCCCCAAGTAATGGCGAATTAAAGGGGA S.siamkayai -TTAAGCGGAGGAAA-GAAACTAACTTAGGATTCCCCAAGTAATGGCGAATGAAAGGGGA S.carpocapsae -TAAGCCGGAGGAAAAGAAACTAACTTAGGATTCCCCAAGTAATGGCGAATGAAAGGGGA SteinD3 --TAGCGGAGGGAAAAGAAACTAA-CTAGGATTCCCCAAGTAATGGCGAATGAAAGGGGA S.scapterisci --TACNNGGAGGAAAAGAAACTAACTTAGGATTCCCCAAGTAATGGCGAATGAAAGGGGA C.elegans ATTTAGCGGAGGAAAAGAAACTAA-AAAGGATTCCCTTAGTAACGGCGAGTGAAACGGGA ** * * * ** * S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 AGAGCTCAGCGTCGAAACCGTATTTAACTTGTTTAGTGCGGTGTTGTGACGTACAGATTC AGAGCTCAGCGTCAAAACCGTATTTAACTTGCTTAGTGCGGTGCTGTGACGTACAGATTC AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT 19

21 20 S.feltiae AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.feltiae5C AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.feltiaeKrK3 AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.feltiaeR03 AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.feltiaeR04 AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.puntauvense AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.kraussei AGAGCTCAGCGTCGAAACCGTGTTGGCTCTCGTTGACACGGTATTGTGACGTATAGAGGT S.oregonense AGAGCTCAGCGTCGAAACCGTGTTGGCTTTCGTTGACACGGTATTGTGACGTATAGAGGT S.kushidai AGAGCTCAGCGTCGAAACCGTGTTGGCTTTTGTTGACATGGTATTGTGACGTATAGAGGT S.diaprepesi AGAGCTCAGCGTCGAAACCGTGTTGGCTTT-GCTGACACTGTACTGTGACGTATAGAGGT S.puertoricense AGAGCTCAGCGTCGAAACCGTGTTGGCTTT-GCTGACACTGTATTGTGACGTATAGAGGC Stein75-2 AGAGCTCAGCGTCGAAACCGTGTCGGCTCT-GCTGACACTGTATTGTGACGTATAGAGGT S.cubanum AGAGCTCAGCGTCGAAACCGTGTTGGCCCT-GTCGACACTGTATTGTGACGTATAGAGGC S.longicaudum AGAGCTCAGCGTCGAAACCGTGTTGGCCCT-GTCGACACTGTATTGTGACGTATAGAGGC S.glaseri AGAGCTCAGCGTCGAAACCGTGTTGGCCCT-GTCGACACTGTATTGTGACGTATAGAGGC S.arenarium AGAGCTCAGCGTCGAAACCGTGTCGGCTCTCGTCGACACTGTATTGTGACGTATAGAGGT S.hermaphroditum AGAGCTCAGCGTCGAAACCGTGTTGGCTCT-GCTGACACTGTATTGTGACGTATAGAGGT S.scarabaei AGAGCTCAGCGTCGAAACCGTGTTGGCTCT-GCTGACACTGTATTGTGACGTATAGAGGT S.khoisanae AGAGCTCAGCGTCGAAACCGTGTTGGCTTT-GCTGACACTGTATTGTGACGTATAGAGGT S.karii AGAGCTCAGCGTCGAAACTGTGCTGGCTTC-GCCGGCACCGTGTTGTGACGTATAGAGGT S.rarum AGAGCTCAGCGTCGAAACCATACTGGCTTT-GCTGATATGGTGTTGTGACGTATAGAGGT S.costaricense AGAGCTCAGCGTCGAAACCGTGTTGACTAGCGTTGACACTGTATTGTGACGTATAGAGGC S.bicornutum CAAGTTCAGCGTCGAAGCTGCGATGGCTAACGTTGACGTGGTATTGTGACGTATAGAGGT S.ceratophorum AAAGTTTAGCGTCTAAACTGCGTTGACTAACGTTGACGTGGTATTGTGACGTATAGAAGT S.abassi ACAGCTCAGCGTCGAAATCACGTTGGCTAACGTTGACGTGGTGTTGTGACGTATAGAGGT S.abbasi-S01-Iran -CAGCTCAGCGTCGAAATCACGTTGGCTAACGTTGACGTGGTGTTGTGACGTATAGAGGT S.riobrave AAGGCTCAGCGTCTAAACCATGCTGGCTAGCGTTGGCATGGTATTGTGACGTAAAGAGGT S.websteri AAAGCTCAGCGTCGAAACCAAGTTGGCTAACGTTGACTTGGTGTTGTGACGTATAGAGGC S.anatoliense AAAGC-CAGCGTCGAAACCAAGTTGGCTAACGTTGACTTGGTGTTGTG-CGTATAGAGGC S.monticolum AGAGCCCAGCGCTGAATC--TCTCGGTCTTAGGCCGCTGAGAACTGTAGCGTATAGGTGT S.siamkayai AGAGCCCAGCGCTGAATC--TCTCGGTCTTAGGCCGCTGAGAACTGTAGCGTATAGGTGT S.carpocapsae AGAGCCCAGCGCTGAATC--TCTCGGTCTTAGGCCGCTGAGAACTGTAGCGTATAGGTGT SteinD3 AGAGCCCAGCGCTGAATC--TCTCGGTCTTAGGCCGCTGAGAACTGTAGCGTATAGGTGT S.scapterisci AGAGCCCAGCGCTGAATC--TCTCGGTCTTAGGCCGCTGAGAACTGTAGCGTATAGGTGT C.elegans AGAGCCCAGCGCCGAATC--GATCAGTCTTTGGCTGCTTCGAAATGTGGCGTATAGGTGT * **** ** * *** **** ** S.affine AACCATGTGCGT-TTTGTTGACAATACGAATTTCCTTT-GACTAGGG-----ATCCAAAG S.intermedium AATCATGCGCGT-TTTGTTGACGTTACGAATTTCCTTT-GACTAGGG-----ATCCAAAG S.feltiae60 GATCATGTGCGG-TTTGCTAGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeB02 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeK4 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeTurS3 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeCK6 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiae GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiae5C GATCATGTGCGG-TTTGCTAGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeKrK3 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeR03 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.feltiaeR04 GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.puntauvense GATCATGTGCGGGTTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.kraussei GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.oregonense GATCATGTGCGG-TTTGCTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.kushidai GATCATGTGCGG-TTTGTTGGCTTTACGAATTTCCTTT-GACTAGGA-----ATCCATAG S.diaprepesi GATCATGTGCGG-TTTACTGGCTTTACGAATTCCCTTT-GACTAGGG-----ATCCAAAG S.puertoricense GATCATGTGCGG-TTTACTGACTTTACGAATTCCCTTT-GACTAGGG-----ATCCAAAG Stein75-2 GATCATGTGCGG-TTTACGGGCTTTACGAATTCTCTTT-GACTAGAG-----ATCCAAAG S.cubanum GGTCATGTGCGT-TTGGCTGGTTGTACGAAATCCCTTT-GACTAGGG-----TTCCAAAG S.longicaudum GGTCATGTGCGT-TTGGCTGGTTGTACGAAATCCCTTT-GACTAGGG-----TTCCAAAG S.glaseri GGTCATGTGCGT-TTGGCTGGTTGTACGAAATCCCTTT-GACTAGGG-----TTCCAAAG S.arenarium GGTCATGTGCGG-GTTACGGACGTTACGAATTCCCTTT-GACTAGGG-----ATCCAAAG S.hermaphroditum GATCATGTGCGG-TTTACTGGCTTCACGAATTCTCTTTTGACTAGAG-----ATCCAAAG S.scarabaei GATCATGTGCGG-TTTACTGGCTTCACGAATTCTCTTT-GACTAGAG-----ATCCAAAG S.khoisanae GATCATGTGCGG-TTTACTGGCTTTACGAATTCCCTTT-GACTAGGG-----ATCCAAAG S.karii GATCATGTGCGG-TTTGCGGACTTTACGAATTCTCTTT-GACTAGGG-----ATCCAAAG S.rarum GTTCATGTGTGA-TTTGTTGGCTATACGAATTTCCTTT-GACCAGGG-----ATCCATAG S.costaricense GTTCATGTGCGT-GTTGTTGGCTTCACAAAATTCCTTT-GACTAGGA-----TTCCATAG S.bicornutum GTTCATGTGCGT-TTAACTGATAATACGAATTTTCTTT-GACTAGAA-----ATCCAAAG

22 S.ceratophorum S.abassi S.abbasi-S01-Iran S.riobrave S.websteri S.anatoliense S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Stein75-2 S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii S.rarum S.costaricense S.bicornutum S.ceratophorum S.abassi S.abbasi-S01-Iran S.riobrave S.websteri S.anatoliense S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C GTACATGTGCGT-TTAGCTGATAATACGAATTTTCTTT-GACTAGAA-----ATCCAAAG GT-TATGTGCGT-TTTGTTGGTGGTACGAATTTACTTT-GACTAGTA-----ATCCAAAG GT-TATGTGCGT-TTTGTTGGTGGTACGAATTTACTTT-GACTAGTA-----ATCCAAAG GTCCATGTGCGT-TTGGCTAGTTGTACGAACTATCCTT-GAATAGAT-----GTCCATAG GTTCATGTGCGG-TTTGTTGATAATGCGAATTTCCTTT-GACTAGGG-----ATCCAAAG GTTCATGTGCGG-TTTGTTGATAATGCGAATTTCCTTTTGACTAGGG-----ATCCAAAG GA--CTGTCCTGCTATATTGCATTTCCGAAGTCTCTTT-GATAGGGGCCAACATCCAGAG GA--CTGTCCTGCTATATTGCATTTCCGAAGTCTCTTT-GATAGGGGCCAACATCCAGAG GA--CTGTCCTGCTATATTGCATTTCCGAAGTCTCTTT-GATAGGGGCCAACATCCAGAG GA--CTGTCCTGCTATATTGCATTTCCGAAGTCTCTTT-GATAGGGGCCAACATCCAGAG GA--CTGTCCTGCTATATTGCATTTCCGAAGTCTCTTT-GATAGGGGCCAACATCCAGAG AA--GTTTCCAGCAGTGTCGTATGTCCGAAGTCCTTAC-GATTGAGGCCATAAACCAGAG * * ** * ** *** ** CGGGTGCTAGACCTGTACGTATTGTTAATTTTGCGTGCGCGTTT-GTTTCTTTGAGTAGG CGGGTGCTAGACCTGTACGTATCGTTAATTTAGCGTGCGCGTTT-GTTTTTTTGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTTTCGTACGCGTTTTATCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGCCGACTTTCTGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGCCGACTTTCTGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGCCGGCTTTTCGTACGCGTTT-ATCTCTTGGAGTAGG CGGGTGCAAGACCCGTACGTATTGCCGGTTTGTCGTACGCGTTT-GCTTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGTTGGTTTGTCGTACGCGTTT-GTCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGCCTGTTTGTCGTACGCGGTT-GCTTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTGCAGCCGGCCGTTCGTACGCGGCA-GTCTTTTGGAGTAGG CGGGTGCGAGACCCGTACGTGCAGCCGGCCGTTCGTACGCGGCA-GTCTTTTGGAGTAGG CGGGTGCGAGACCCGTACGTGCAGCCGGTCGTTCGTACGCGGCT-ACCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATCGTCGGTTTTTCGTACGCGGCC-GCCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTGTTGCCGGTTTGTCGTACGCGTTTTGCTTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTGTTGCCGGTTTGTCGTACGCGTTT-GCTTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGCCGGTTTGTCGTACGCGTTT-GCTTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTATTGTCTGCTTGTCGTACGCGTTT-GCTTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTACAGCTGACTTATTGTACGCGTTC-ATCTCTTTGAGTAGG CGGGTGCGAGACCCGTACGTGTCGCTGGCTTTTCGTACGCGTTC-GTCTCTTGGAGTAGG CGGGTGCGAGACCCGTACGTGTTGTTGGTTTTTCGTACGCGTTC-ATCTCTTGGAGTAGG CTGGTGCCAGACCCGTACGTGTTGTTGGCTTTTCGTACGCGTTC-ATTTCTTGGAGTAGG CGGGTGCGAGACCTGTACGTGTTGTTGACTTAATGTGCGCTTGC-ACTTTTTTGAGTAGG CGGGTGCGAGACCTGTACGTGTTGTTGACTTAATGTGCGCTTGC-ACTTTTTTGAGTAGG TGGGTGCAAGACCTGTACGTGCAGCTGGCTTTTCGTACGCGTTC-ATTTCTTGGAGTAGG AGGGTGCTAGACCCTTACGCATTGTTGACTTTTCGTACGCGTTC-GTTTCTTGGAGTAGG AGGGTGCTAGACCCTTACGCATTGTTGACTTTTCGTACGCGTTC-GTTTCTTGGAGTAGG AGGGTGCGAGACCTGTAC-GGAAAGTAGTATAGTTGGTATGGTT-ACTCCTTGGAGTCGG AGGGTGCGAGACCTGTAC-GGAAAGTAGTATAGTTGGTATGGTT-ACTCCTTGGAGTCGG AGGGTGCGAGACCTGTAC-GGAAAGTAGTATAGTTGGTATGGTT-ACTCCTTGGAGTCGG AGGGTGCGAGACCTGTAC-GGAAAGTAGTATAGTTGGTATGGTT-ACTCCTTGGAGTCGG AGGGTGCGAGACCTGTAC-GGAAAGTAGTATAGTTGGTATGGTT-ACTCCTTGGAGTCGG AGGGTGCGAGCCCCGTTCTGGATAGCGGCACTGTTGGTTCGCTT-GCTCCTTGGAGTCGG ***** ** ** * * ** **** ** GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC 21

23 22 S.feltiaeKrK3 GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.feltiaeR03 GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.feltiaeR04 GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.puntauvense GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.kraussei GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.oregonense GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.kushidai GTTGTTTGAGATCGCAGCCCTAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.diaprepesi GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.puertoricense GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC Stein75-2 GTTGTTTGAGATCGCAGCCCAAAGCAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.cubanum GTTGTTTGAGATCGCAGCCCAAAGCAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.longicaudum GTTGTTTGAGATCGCAGCCCAAAGCAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.glaseri GTTGTTTGAGATCGCAGCCCAAAGCAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.arenarium GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.hermaphroditum GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.scarabaei GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.khoisanae GTTGTTTGAGATCGCAGCCCAAAGAAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.karii GTTGTTTGAGATCGCAGCCCAAAGAAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.rarum GTTGTTTGAGATCGCAGCCCGAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.costaricense GTTGTTTGAGATCGCACGCCCAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.bicornutum GTTGTTTGAGATCGCAGCTCGAAGTAGG-TGGTATACTTCATCTAAA-ACTAAATATGAC S.ceratophorum GTTGTTTGAGATCGCAGCTCGAAGTAGG-TGGTATACTTCATCTAAA-ACTAAATATGAC S.abassi ATTGTTTGAGATCGCAGTCCGAAGCAGG-TGGTATACTTCATCTAAA-GCTAAATATGAC S.abbasi-S01-Iran ATTGTTTGAGATCGCAGTCCGAAGCAGG-TGGTATACTTCATCTAAA-GCTAAATATGAC S.riobrave GTTGTTTGAGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATATGAC S.websteri GTTGTTTTGGATCGCAGCCCAAAGTAGG-TGGTATACTTCATCTAAA-GCTAAATACGAC S.anatoliense GTTGTTTTGGATCGCAGCCCAAAGTAGGGTGGTATACTTCATCTAAAAGCTAAATACGAC S.monticolum GTTGCTTGAAAGTGCAGCCTAAAGTTGG-TGATAAACTTCATCTAAG-GCTAAATATCGA S.siamkayai GTTGCTTGAAAGTGCAGCCTAAAGTTGG-TGATAAACTTCATCTAAG-GCTAAATATCGA S.carpocapsae GTTGCTTGAAAGTGCAGCCTAAAGTTGG-TGATAAACTTCATCTAAG-GCTAAATATCGA SteinD3 GTTGCTTGAAAGTGCAGCCTAAAGTTGG-TGATAAACTTCATCTAAG-GCTAAATATCGA S.scapterisci GTTGCTTGAAAGTGCAGCCTAAAGTTGG-TGATAAACTTCATCTAAG-GCTAAATATCGA C.elegans GTTGCTTGAAAGTGCAGCCTAAAGTGGG-TGATAAACTTCATCTAAG-GCTAAATATCGA *** ** * *** *** ** ** ** *********** ******* S.affine TACGAATCCGATAGTAAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA S.intermedium TACGAATCCGATAGTAAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Stein75-2 S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii TACGAATCCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGAACTTTGAAGAGAGA S.rarum TATGAATCCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA S.costaricense TACGAATCCGATAGCAAACAAGTACCGTGAGGGAAAATTGCAAAGTACTTTGAAGAGAGA S.bicornutum TATGAATCCGATAGCGAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA S.ceratophorum TACGAATCCGATAGCGAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA S.abassi TACGAATCCGATAGCGAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA

24 S.abbasi-S01-Iran S.riobrave S.websteri S.anatoliense S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Stein75-2 S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii S.rarum S.costaricense S.bicornutum S.ceratophorum S.abassi S.abbasi-S01-Iran S.riobrave S.websteri S.anatoliense S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 TACGAATCCGATAGCGAACAAGTACCGTGAGGGAAAGTTGCAAAGTACTTTGAAGAGAGA CGCGAAATCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGAACTTTGAAGAGAGA CGCGAAATCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGAACTTTGAAGAGAGA CGCGAAATCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGAACTTTGAAGAGAGA CGCGAAATCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGAACTTTGAAGAGAGA CGCGAAATCGATAGCAAACAAGTACCGTGAGGGAAAGTTGCAAAGAACTTTGAAGAGAGA CTCGATTGCGATAGCGAACAAGTACCGTGAGGGAAAGTTGCAAAGGACTTTGAAGAGAGA ** ****** ******************** ******** ************** GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACAGG GTTCAAGAGGACGTGAAACCGATAGGGTGGAAGCAGATGAAGTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGATAGGATGGAAGCAGATGAAGTTGACG------AACGAG GTTAAAAAGGACGTGAAACCGGTAGGGTGGAAGCAGATGAAGTTAACG------AACGAG GTTAAAAAGGACGTGAAACCGGTAGGATGGAAGCAGACAAAGTTAACG------AACGAG GTTCAAAAGGACGTGAAACCGATAGGATGGAAGCAGATAAAATTGACG------AACGGG GTTCAAAAGGACGTGAAACCGATAGGATGGAAGCAGATAAAATTGACG------AACGGG GTTCAAGAGGACGTGAAACCGATAGGGTGGAAGCAAATGAGTTTGACG------AACGAG GTTCAAGAGGACGTGAAACCGGTAGGGTGGAAGCAGATAAAGTTGACG------AACGTG GTTCAAGAGGACGTGAAACCGGTAGGGTGGAAGCAGATAAAGTT-ACG------AACGTG GTTCAAGAGAACGTGAAATCGCTAAAGTGGAACCGGAGAGAGTTAACATAACTTGGTAGC GTTCAAGAGAACGTGAAATCGCTAAAGTGGAACCGGAGAGAGTTAACATAACTTGGTAGC GTTCAAGAGAACGTGAAATCGCTAAAGTGGAACCGGAGAGAGTTAACATAACTTGGTAGC GTTCAAGAGAACGTGAAATCGCTGAAGTGGAACCGGAGAGAGTTAACATAACTTGGTAGC GTTCAAGAGAACGTGAAATCGCTAAAGTGGAACCGGAGAGAGTTAACATAACTTGGTAGC GTTCAAGAGAACGTGAAATCGCTGGAGTGGAACCGGAGACAGTTGATGTTGCTTGGAGAC *** ** ** ******** ** * ***** * * ** * TGTTGTATTCAG-AGTTACAT--TTATG--TGTAATTTGTTTTTGCGACCAAT--GTGGG CGTTGTATTCAG-AGTTACAT--TTTTG--TGTAATTTGTTTTTGCGACCAAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG 23

25 24 S.feltiaeR04 TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG S.puntauvense TGTCGTATTCAA-AATCGC-----TGTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG S.kraussei TGTCGTATTCAG-AATCGC-----TTTC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG S.oregonense TGTCGTATTCAG-AATCGC-----TATC--AGCGGTTTGTTTTTACGGCCGAT--GTGGG S.kushidai TGTCGTATTCAG-AATCGC-----TGTC--AGCGGTTTGTTCTTACGACCGAT--GTGGG S.diaprepesi TGTCGTATTCAG-GGTTGCATC-TTGTG--TGCGGTCTGTTCTTGCGACCGAT--GTGGG S.puertoricense TGTCGTATTCAG-GGTTGCATC-TTGTG--TGCGGTCTGTTCTTGCGACCGAT--GTGGG Stein75-2 TGTCGTATTCAG-GGTTGCATC-TTGTG--TGCAGTCTGTTCTTGCGGCCGAT--GTGGG S.cubanum TGTCGTATTCAG-GGTCGCGTC-TTGTG--CGCGGTCTGTTCTTGCGACCGAC--GTGGG S.longicaudum TGTCGTATTCAG-GGTCGCGTC-TTGTG--CGCGGTCTGTTCTTGCGACCGAC--GTGGG S.glaseri TGTCGTATTCAG-GGCCGCGTC-TTGTG--CGCGGTCTGTTCTTGCGACCGAC--GTGGG S.arenarium TGTCGTATTCAG-GGCCGCATC-TTGTG--TGCGGTCTGTTCTTGCGACCGAT--GTGGG S.hermaphroditum TGTCGTATTCAG-GGTTGCATC-TTGTG--TGCAGTCTGTTCTTGCGGCCGAT--GTGGG S.scarabaei TGTCGTATTCAG-GGTTGCATC-TTGTG--TGCAGTCTGTTCTTGCGGCCGAT--GTGGG S.khoisanae TGTCGTATTCAG-GGTTGCATC-TTGTG--TGCGGTCTGTTCTTGCGACCGAT--GTGGG S.karii CGCCGTATTCAG-GGCTGTGCC-TCGTG--TGCAGTCTGTTTTTGCGGCCGAT--GTGGG S.rarum TGTTGTACTCAG-AATTACATC-TTGTG--TGTAGTTTGTTCTTGCAACCGAT--GTGGG S.costaricense TGTCGTATTCAG-AATCGTATC-TTGTG--TGCGGTTTGTTCTTGCGACCGAT--GTGGG S.bicornutum CGTTGTATTCAG-AATCGTATC-TTGTG--TACGGTTTGTTTTTACAGCCGAT--GTGGG S.ceratophorum TGTTGTATTCAA-AATCGTATC-TTGTG--TACGGTTTGTTCTTACAGCCGAT--GTGGG S.abassi TGTCGTATTCAG-GATTGTATC-TTGTG--TGCAGTTTGTTTTTACGGCCGTT--GTGGG S.abbasi-S01-Iran TGTCGTATTCAG-GATTGTATC-TTGTG--TGCAGTTTGTTTTTACGGCCGTT--GTGGG S.riobrave TGTCGTATTCAG-GGCCGTATC-TTGTG--TACGGTCTGTTTTTACGGCCG-T--GTGGG S.websteri TGTCGTATTCAG-AACTACA-----ATT--TGTGGTTTGTTTTTACGATCGAT--GTGGG S.anatoliense TGTCGTATTCAG-AACTACA-----ATT--TGTGGTTTGTTTTTACGATCGAT--GTGGG S.monticolum TATATTCTCTAA-AGTCGCTTAATTGTGGCTCTGGGGGGTGGACGCTACCTGTTGTTGCA S.siamkayai TATATTCTCTAA-AGTCGCTTAATTGTGGCTCTGGGGGGTGGACGCTACCTGTTGTTGCA S.carpocapsae TATATTCTCTAA-AGTCGCTTAATTGTGGCTCTGGGGGGTGGACGCTACCTGTTGTTGCA SteinD3 TATATCCTCTAA-AGTCGCTTAACTGTGGCTCTGGGGGGTGGACGCTACCTGTTGTTGCA S.scapterisci TATATTCTCTAA-AGTCGCTTAATTGTGGCTCTGGGGGGTGGACGCTACCTGTTGTTGCA C.elegans AAGCTTGGTGACTGGTCGCTTAGTTGTGATCGTTGCCGGGTGTCGTTTCCTAT-GCTACG * * * * * S.affine CTGACGTATTCGGTTAATGCGAT--GTGCTTATCAATGATGGCGGCCCTGCGGAGG--GA S.intermedium CTGACGTATTCGGTTAATGCGAT--GTGCTCAGCAATGATGGCGGCCCTGCGGAGG--GA S.feltiae60 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeB02 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeK4 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeTurS3 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeCK6 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiae CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiae5C CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeKrK3 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeR03 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.feltiaeR04 CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.puntauvense CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCGGAGG--GA S.kraussei CTGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGTGACCCTGCAGAGG--GA S.oregonense CCGGCGGCTTTGGTCAATGCAAC--GCGTCTAGCGTCGATGGAGACCCTGCGGAGG--GA S.kushidai CTGGCGGCTTTGGTCAATGTAAT--GTGTCTAGCGTCGATGGAGACCCTGCGGAGG--GA S.diaprepesi CTGGCGGCTTTGGTCAATGCACT--GCGTCAAACGTCTATGGAGACCCTGCGGAGG--GA S.puertoricense CTGGCGGCTTTGGTCAATGCACT--GTGCCAAGCGTCTATGGAGACCCTGCGGAGG--GA Stein75-2 CCGGCGGCTTTGGTCAATGCACT--GTGTCAAGCGTCGATGGTGACCCTGCGGAGG--GA S.cubanum CCGGCGGTCTTGGTCAATGCACT--GTGTCTAGCGTCGATGGAGACCCTGCGGAGG--GA S.longicaudum CCGGCGGTCTTGGTCAATGCACT--GTGTCTAGCGTCGATGGAGACCCTGCGGAGG--GA S.glaseri CCGGCGGTCTTGGTCAATGCACT--GTGTCTAGCGTCGATGGAGACCCTGCGGAGG--GA S.arenarium CTGGCGGCCTTGGTCAATGCACTCTGTGTCAAGCGTCGATGGTGACCCTGCGGAGG--GA S.hermaphroditum CTGGCGGCTCTGGTCAATGTGCT--GTGTCAAGCGTCGATGGTGACCCTGCGGAGG--GA S.scarabaei CTGGCGGCTCTGGTCAATGTGCT--GTGTCAAGCGTCGATGGTGACCCTGCGGAGG--GA S.khoisanae CCGGCGGCCTTGGTCAATGCACT--GTGTCAAGCGTCGATGGAGACCCTGCGGAGG--GA S.karii CCGGCGGTCTTGGTCAATGCACT--GTGTCAAGCGGCGATGGAGACCCTGCGGAGG--GA S.rarum CTGGCGTGCTTGGTCAATGCATT--GTGTCAAGCGTCGATGGTGACCCTGCGGAGG--GA S.costaricense CTGACGACTCTGGTTAATGCGCT--GTGTTTAGCGTCGATGGCGACTCTGCGGAGG---A S.bicornutum CTGGCGGCTTTGGTCAATACAAT--GTGTTTGGCGTCGATGGTGACCCTGAGGAGG--GA S.ceratophorum CTGGCGGCTTTGGTCAATACAAT--GTGTTTGGCGTCGATGGTGACCCTGCGGAGG--GA S.abassi CTGGCGGCTTTGGTCAATGCAAT--GTGTTTGGCATTGAGAGTGACCCTGCGGAGG--GA S.abbasi-S01-Iran CTGGCGGCTTTGGTCAATGCAAT--GTGTTTGGCATTGAGAGTGACCCTGCGGAGG--GA S.riobrave CTGGCGGCTTTGGTTAATGTAA---GTGTTTGGCATTGATGGTGACCCTGTGGGGG--GA

26 25 S.websteri CTGGCGTCTTTGGTTAACTTAGT----GTCTGGCGGCAATGGTGACCCTGCGGAGG--GA S.anatoliense CTGGCGTCTTTGGTTAACTTAGT----GTCTGGCGGCAATGGTGACCCTGCGGAGG--GA S.monticolum CTGACTGCGTTGATTG-TGCTTTCCGAACAGTATAGTGTTGCCCACTTTGT GG S.siamkayai CTGACTGCGTTGATTG-TGCTTTCCGAACAGTATAGTGTTGCCCACTTTGT GG S.carpocapsae CTGACTGCGTTGATTG-TGCTTTCCGAACAGTATAGTGTTGCCCACTTTGT GG SteinD3 CTGACTGCGTTGATTG-TGCTTTCCGAACAGTATAGTGTTGCCCACTTTGT GG S.scapterisci CTGACTGCGTTGATTG-TGCTTTCCGAACAGTATAGTGTTGCCCACTTTGT GG C.elegans CCGACGGCGTTGGCTG-CTCGTTCTAGCCCG-ACAGTGTTGCCCATCTCGCAAGAGAAGG * * * * * S.affine TTACCAATCAG-TGTACGGTGCTCGGTATAGCTGGATGTTT------T S.intermedium TTACCAATCAG-TGTACGGTGCTCGGTATAGCTGAATGTTT------T S.feltiae60 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeB02 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeK4 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeTurS3 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeCK6 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiae CAATCAGTCGT-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiae5C CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeKrK3 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeR03 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.feltiaeR04 CAATCAGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.puntauvense CAATCAGTCGT-CGTACGGTGCTTGGTATGGCTAAGGTTT S.kraussei TAATCAATCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.oregonense CAATCGGTCGG-CGTACGGTGCTTGGTATGGCTAAGGTTT S.kushidai TAATCAGTCGG-CGTACGGTGCTTAGTATGGCTAAGGTTT S.diaprepesi TCATCAGTGGG-CTTGCGATGCGTGGTATGGCTAAGGTTT S.puertoricense TCATCAGTAGG-CGTACGGTGCGTGGTATGGCTAAGGTTT Stein75-2 TAATCGGTCGG-CGTGCGATGCGTGGTATGGCTAGGGTG S.cubanum TCATCAGTCGG-CGTACGATGCGTGGTATGGCTAAGGTTCTG-----T S.longicaudum TCATCAGTCGG-CGTACGATGCGTGGTATGGCTAAGGTTCTG-----T S.glaseri TCATCAGTCGG-CGTACGATGCGTGGTATGGCTAAGGTTCTG-----T S.arenarium CCGTCGGTCGG-CGTACGATGCGTGGTATGGCTAAGGTTTG S.hermaphroditum TAATCAGTCGGTCGTACGATGCGTAGTATGGCTAGAGTTT S.scarabaei TAATCAGTCGGTCGTACGATGCGTAGTATGGCTAGAGTTT S.khoisanae TAATCATTCGG-CGTGCGATGCGTGGTATGGCTAAGGTTTT S.karii TCATCAGTCGT-CGTACGATGCGTGGTATGGCTAAGCGCTT S.rarum TACTCGTTCGG-CGTAAGGTGCGTGGTATGGCTAAGTAGT S.costaricense CAATVAGTCGA-TGTGCGATGCGTGGTATAGCTAGGATTT S.bicornutum TATTCGTTCGG-TGTGCGATGCTTGATATGGCTAAGGTAT S.ceratophorum AATTCGGTCGG-CGTGCAATGCTTGATATGACTAAGGTTT S.abassi TATTCGCTCAG-TGTGCGGTGCTTGGTATGGCTAAGGTAT S.abbasi-S01-Iran TATTCGCTCAG-TGTGCGGTGCTTGGTATGGCTAAGGTAT S.riobrave AATTCGTTCGA-TGTGCGATGCTTAGTATAGCTAAGGTTT S.websteri TACTCGGTTGT-CGTGCGATGCTTGGTATGGCTAGAGGTT S.anatoliense TACTCGGTTGT-CGTGCGATGCTTGGTATGGCTAGAGGTT S.monticolum TGTCTTGCTTTC--TGGGAGGTTTGTCATAATTAACGTAAAGCCA----ATTCCCTTTGG S.siamkayai TGTCTTGCTTTC--TGGGAGGTTTGTCATAATTAACGTAAAGCCA----ATTCCCTTTGG S.carpocapsae TGTCTTGCTTTC--TGGGAGGTTTGTCATAATTAACGTAAAGCCA----ATTCCCTTTGG SteinD3 TGTCTTGCTTTC--TGGGAGGTTTGTCATAATTAACGTAAAGCCA----ATTCCCTTTGG S.scapterisci TGTCTTGCTTTC--TGGGAGGTTTGTCATAATTAACGTAAAGCCA----ATTCCCTTTGG C.elegans TGTCTTGCTGGCGGTAGTGGGTTCGTGGCGGCTAGCGTTTAGTTACGCTAGTGTGTGTGA * * * S.affine CGTCGGTCTCAAA-GTCAATGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.intermedium CGTCGGTCTTAAA-GTCAATGCCTTATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiae60 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeB02 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeK4 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeTurS3 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeCK6 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiae CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiae5C CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeKrK3 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeR03 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.feltiaeR04 CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.puntauvense CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT

27 26 S.kraussei CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.oregonense CGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.kushidai TGCCGGTCTTAAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.diaprepesi CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.puertoricense CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT Stein75-2 CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.cubanum CGCCGGTCTTGAAAGTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.longicaudum CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.glaseri CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.arenarium CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.hermaphroditum CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.scarabaei CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.khoisanae CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.karii CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.rarum CGCCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.costaricense CGTCGGTCTTGAA-GTCAGCGCCTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.bicornutum CGCTGGTCTTGAA-GTTATTGCTTCATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.ceratophorum CGCCGGTCTTGAA-GTTATTGCTTTTTCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.abassi CGCCGGTCTTGAA-GTCAGAGATTTATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.abbasi-S01-Iran CGCCGGTCTTGAA-GTCAGAGATTTATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.riobrave CGCCGGTCTTGAA-GTCAATTCCTCATTTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.websteri CGCTGGTTTTATA-GTCATCGCTTTATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.anatoliense CGCTGGTTTTATA-GTCATCGCTTTATCTGACCCGTCTTGAAACACGGACCAAGGAGTGT S.monticolum AGTCAGTGTGAAA-GTTAACCACCTTTCCGACCCGTCTTGAAACACGGACCGAGGAGTGT S.siamkayai AGTCAGTGTGAAA-GTTAACCACCTTTCCGACCCGTCTTGAAACACGGACCGAGGAGTGT S.carpocapsae AGTCAGTGTGAAA-GTTAACCACCTTTCCGACCCGTCTTGAAACACGGACCGAGGAGTGT SteinD3 AGTCAGTGTGAAA-GTTAACCACCTTTCCGACCCGTCTTGAAACACGGACCGAGGAGTGT S.scapterisci AGTCAGTGTGAAA-GTTAACCACCTTTCCGACCCGTCTTGAAACACGGACCGAGGAGTGT C.elegans CGTCGGTGTGAAA-GTCGACGACGTTTCCGACCCGTCTTGAAACACGGATTGCGGAGTGC * ** * * ** * ******************** ****** S.affine AGC-GTTTACGCA-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGTAACGAAAGTAAAGAT S.intermedium AGC-GTTTACGCA-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGTAACGAAAGTAAAGAT S.feltiae60 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeB02 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeK4 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeTurS3 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeCK6 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiae AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiae5C AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeKrK3 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeR03 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.feltiaeR04 AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.puntauvense AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.kraussei AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGAAACGAAAGTAAATGC S.oregonense AGC-GTTTGCGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGAAACGAAAGTAAATGC S.kushidai AGC-GTTTGCGCA-AGTCTTAGAGTGTGTCGAAACTTTGAGGCGTAACGAAAGTAAATGC S.diaprepesi AGC-GTTTACGCG-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTAAAGGT S.puertoricense AGC-GTTTACGCA-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTAAAGGT Stein75-2 ATC-GTTTACGCA-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTGAATGT S.cubanum AGC-GTTTACGCG-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTAAAGGT S.longicaudum AGC-GTTTACGCG-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTAAAGGT S.glaseri AGC-GTTTACGCG-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTAAAGGT S.arenarium AGC-GTTTACGCA-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAACGAAAGTAAAGGT S.hermaphroditum AGC-GTTTACGCA-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGCAACGAAAGTGAATGC S.scarabaei AGC-GTTTACGCA-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGCAACGAAAGTGAATGC S.khoisanae AGC-GTTTACGCA-AGTCTTAGAGTGTATCAAAACTTTGAGGCGCAATGAAAGTAAATGC S.karii ATC-GTTTACGCA-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGAAACGAAAGTGAATGC S.rarum AGC-GTTTACGCA-AGTCTTAGAGTGTATCGAAACTTTGAGGCGTAACGAAAGTAAAGGC S.costaricense AGCCGTTTGCGCA-AGTCTTTGAGTGTGTCGAAACTTACAGGCGTAACGAAAGTGAATTC S.bicornutum AGC-GTTTACGCA-AGTCTTAGAGTGTGTCGAAACTTTGAGGCGTAACGAAAGTGAATGT S.ceratophorum AGC-GTTTATGCG-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGTAACGAAAGTGAATGT S.abassi AGC-GTTTGCGCA-AGTCTTAGAGTGTGTCGAAACTTTGAGGCGTAACGAAAGTGAATAT S.abbasi-S01-Iran AGC-GTTTGCGCA-AGTCTTAGAGTGTGTCGAAACTTTGAGGCGTAACGAAAGTGAATAT S.riobrave ATC-GTTTACGCA-AGTCTTAGAGTGTGTGAAAACTTAGAGGCGGAACGAAAGTGAATGT S.websteri ACC-GCTTACGCG-AGTCTTAGAGTGTGTCAAAACTTTGAGGCGTAACGAAAGTAAATGT S.anatoliense ACC-GCTTACGCGGAGTCTTAGAGTGTGTCAAAACTTTGAGGCGTAACGAAAGTAAATGT

28 S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Stein75-2 S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii S.rarum S.costaricense S.bicornutum S.ceratophorum S.abassi S.abbasi-S01-Iran S.riobrave S.websteri S.anatoliense S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense AAT-TTATACGCG-AGTCAAGGGGTGGTGAAAACCCTAGGGGCGGAATGAAAGTAAAGGC AAT-TTATACGCG-AGTCAAGGGGTGGTGAAAACCCTAGGGGCGGAATGAAAGTAAAGGC AAT-TTATACGCG-AGTCAAGGGGTGGTGAAAACCCTAGGGGCGGAATGAAAGTAAAGGC AAT-TTATACGCG-AGTCAAGGGGTGGTGAAAACCCTAGGGGCGGAATGAAAGTAAAGGC AAT-TTATACGCG-AGTCAAGGGGTGGTGAAAACCCTAGGGGCGGAATGAAAGTAAAGGC TTG-TCTACTGCG-AGTCAAAGGGTGTTAAAA--CCTTGCGGCGAAATGAAAGTAAAGGT ** **** * *** * * * **** ** ****** ** AGCTT--CGGGTTATTAACATGGGATGC-GTTGTTTCGT-GTAAACGGCGTC-GGACTAT AGCTT--CGGGTTATTAACATGGGATGC-GCTGTTTCGT-GTAAACGGCGTC-GGACTAT AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTC-TTGTGGACGGCGCTTGGACCAA AGTTT---A-TCTGCTGACTTGGGATGC-ATTGTCTC-TTGTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTCCTTGTGGACGGCGCT-GGACCAA AGTTT---GATCTGCTGACTTGGGATAC-GTTGTCTC-TTGTGGACGGCGTT-GGACCAT AGCTT---CAGCTGCTGACTTGGGATGC-GTTGTCTCTT-GTGGACGGCGCT-GGACCAT AGCTT---TAGCTGCTGACTTGGGATGC-GTTGTCTCTT-GTGGACGGCGCT-GGACCAT AGCTC---GATCTACTGACTTGGGATGC-GTTGTCTCCT-GTGGACAGCGCT-GGACCAT GACGT--AAGTT-GCTGACTTGGGATGC-GCTGTCTTCTTGTGGACGGCGCT-GGACCAT GACGT--AAGTTTGCTGACTTGGGATGC-GCTGTCTTCT-GTGGACGGCGCT-GGACCAT GACGT--CAAGTTGCTGACTTGGGATGC-GCTGTCTCCT-GTGGACGGCGCT-GGACCAT GGCTTC-GAAGCTGCTGACTTGGGATGC-GCTGTCTCCT-GTGGACGGCGCT-GGACCAT AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTCCT-GTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTCCT-GTGGACGGCGCT-GGACCAA AGTTT---AATCTGCTGACTTGGGATAC-GTTGTCTCCT-GTGGACGGCGTT-GGACCAT AGTTT---AATCTGCTGACTTGGGATGC-GTTGTCTTTT-GTGGACGGCGCT-GGACCAT AATTT---AATTTGCTGAATTGGGATGC-GTCATCTC-TTGTGGATGGCGTC-GGACCAT AACTT---GATTTGATGATTTGGGATAC-GTTGTCTTCTTGTGGACCGGCGTGGGACCAT GGTTT---AATCTACTGAATTGGGATGC-GTTGTCTC-TTGTGGACGGCGTC-GGACCAT GGTTT---AATCTACTGAATTGGGATAC-GTTGTCTC-TTGTGGACGACGTC-GGACCAT AGCTT---GATCTATTAACTTGGGATGC-GTTGTCTC-TTGTGGACGATGTT-GGACCAT AGCTT---GATCTATTAACTTGGGATGC-GTTGTCTC-TTGTGGACGATGTT-GGACCAT GGTTT---AATCCGCTGAATTGGGATAC-GTTGTCTC-TTGTAGACGGCGTC-GGACCAT GGATT---TATTCACTGACTTGGGATAC-GTTGTCTT-TTTTGGATAGCGTT-GGACCAT GGATT---TATTCACTGACTTGGGATACCGTTGTCTT-TTTTGGATAGCGTT-GGACCAT CGGTT---AACCGGTTGACATGGGATCC-GC--TCTT----CGGAGCG--GC-GCACCAT CGGTT---AACCGGTTGACATGGGATCC-GC--TCTT----CGGAGCG--GC-GCACCAT CGGTT---AACCGGTTGACATGGGATCC-GC--TCTT----CGGAGCG--GC-GCACCAT CGGTT---AACCGGTTGACATGGGATCC-GC--TCTT----CGGAGCG--GC-GCACCAT CGGTT---AACCGGTTGACATGGGATCC-GC--TCTT----CGGAGCG--GC-GCACCAT CAGTCTCGAATTGGCCGACGTGGGATCT-GTGTTCTT----CGGAGTGCAGC-GCACCAC * ****** * * * * ** * GGTTCTGTCGTGATTGCTTGCAATGCGGCGGAAAAAGAGCGTAAACGGTGCGACCCGAAA GGTTCTGTCGTGATTGCTTGCAGTGCGGCGGAAAAAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA GGTTTTATCGCGATCGCTTGCGGTGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA 27

29 28 S.kushidai GGTTTTATCGCGATCGCTTGCGATGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA S.diaprepesi GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.puertoricense GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA Stein75-2 AGTTATATCGCGATCGCTTGCGATGCGGTGTAAACAGAGCGTAAGCGGTGCGACCCGAAA S.cubanum GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.longicaudum GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.glaseri GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.arenarium GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.hermaphroditum GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAGCGGTGCGACCCGAAA S.scarabaei GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAGCGGTGCGACCCGAAA S.khoisanae GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.karii GGTTATATCGCGATCGCTTGCGATGCGGTGTAAATAGAGCGTAAACGGTGCGACCCGAAA S.rarum AGTTTTATGGCGATTGCTTGCAATGCTGTGAAAATAGAGCGTAAATGATGCGACCCGAAA S.costaricense AGTTGTATTGCGATTGCTTGCAATGCGGTGCAAATAGAGCGTAAACGGTGCGACCCGAAA S.bicornutum GGTTTTATCGTGATCGCTTGCGATGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA S.ceratophorum GGTTTTATCGTGATCGCTTGCGATGCGGTGAAAATAGAGCGTAAACGGTGCGACCCGAAA S.abassi GGTTTTATCGCGATTGCTTGCAATGCGGTGAAAATTGAGCGCAAATGGTGCGACCCGAAA S.abbasi-S01-Iran GGTTTTATCGCGATTGCTTGCAATGCGGTGAAAATTGAGCGCAAATGGTGCGACCCGAAA S.riobrave GGTTTTATCGCGATCGCTTGCGATGCGGTGAAAATTGAGCGTAGACGGTGCGACCCGAAA S.websteri GGTTTTATCGTAATCGCTTGCGATGCGGTGAAAATAGAGCGTAAGCGGTGCGACCCGAAA S.anatoliense GGTTTTATCGTAATCGCTTGCGATGCGGTGAAAATAGAGCGTAAGCGGTGCGACCCGAAA S.monticolum GGCCCTGTCTTGTCTGCTTGCAGATGGGCAGCGGTAGAGCGTATAGTTTGCGACCCGAAA S.siamkayai GGCCCTGTCTTGTCTGCTTGCAGATGGGCAGCGGTAGAGCGTATAGTTTGCGACCCGAAA S.carpocapsae GGCCCTGTCTTGTCTGCTTGCAGATGGGCAGCGGTAGAGCGTATAGTTTGCGACCCGAAA SteinD3 GGCCCTGTCTTGTCTGCTTGCAGATGGGCAGCGGTAGAGCGTATAGTTTGCGACCCGAAA S.scapterisci GGCCCTGTCTTGTCTGCTTGCAGATGGGCAGCGGTAGAGCGTATAGTTTGCGACCCGAAA C.elegans GGCCCTGTGCGTGTCACTTGTGACTGTGCAGAGGTTGAGCAGTTGGCAAACGACCCGAAA * * * **** * **** ********** S.affine GATGGTGAACTATGCTTGAGCAGGATGAAGCCGGAGGAAACTC-TGGTGGAAGTCC-GAA S.intermedium GATGGTGAACTATGCTTGAGCAGGATGAAGCCGGAGGAAACTC-TGGTGGAAGTCC-GAA S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Stein75-2 S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii S.rarum S.costaricense S.bicornutum S.ceratophorum S.abassi S.abbasi-S01-Iran S.riobrave GATGGTGAACTATGCCTGAACAGGATGAAGCCGGAGGAAACTC-TGGTGGAAGTCC-GAA S.websteri S.anatoliense GATGGTGAACTATGCCTGAGCAGGATGAAGCCGGAGGAAACTCCTGGTGGAAGTCCCGAA S.monticolum GATGGTGAACTATGCTTGAGCAGGACGAAGCCAGAGGAAACTC-TGGTGGAAGTCC-GTA S.siamkayai GATGGTGAACTATGCTTAAACAAGACGAAGCCAGAGGAAACTC-TGGTGGAAGTCC-GTA

30 S.carpocapsae SteinD3 S.scapterisci C.elegans S.affine S.intermedium S.feltiae60 S.feltiaeB02 S.feltiaeK4 S.feltiaeTurS3 S.feltiaeCK6 S.feltiae S.feltiae5C S.feltiaeKrK3 S.feltiaeR03 S.feltiaeR04 S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Stein75-2 S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii S.rarum S.costaricense S.bicornutum S.ceratophorum S.abassi S.abbasi-S01-Iran S.riobrave S.websteri S.anatoliense S.monticolum S.siamkayai S.carpocapsae SteinD3 S.scapterisci C.elegans GATGGTGAACTATGCTTGAGCAGGACGAAGCCAGAGGAAACTC-TGGTGGAAGTCC-GTA GATGGTGAACTATGCTTGAGCAGGACGAAGCCAGAGGAAACTC-TGGTGGAAGTCC-GTA GATGGTGAACTATGCTTGAGCAGGACGAAGCCAGAGGAAACTC-TGGTGGAAGTCC-GTA GATGGTGAACTATGCCTGAGCAGGATGAAGCCAGAGGAAACTC-TGGTGGAAGTCC-GTA *************** * * ** ** ****** ********** ************ * * ACGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA ACGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGG GCGGTTCTGACGTGCAAATCGATCGTCTGAC-T-GGGT GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGGAAGACAA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-T GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGT GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCG- GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCG-TTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGR GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-T-GGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAT-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGAC-TTGGGTATAGGGGCGAAAGACTAATCGA GCGGTTCTGACGTGCAAATCGATCGTCTGGTGTTGGGTATAGGGGCGAAAGACTAATCGA TCGGTTCTGACGTGCAAATCGATCGATAGAC-TTGGGTATAGGGGCGAAAGACTAATCGA TCGGTTCTGACGTGCAAATCGATCGATAGAC-TTGGGTATAAGGGCGAAGGACTAATCGA TCGGTTCTGACGTGCAAATCGATCGATAGAC-TTGGGTATAAGGGCGAAAGACTAATCCA TCGGTTCTGACGTGCAAATCGATCGATAGAC-CT----ATAGGGGCGAAAGACTAATCGA TCGGTTCTGACGTGCAAATCGATCGATAGAC-TTGGGTATACGGGCGAAAGACTAATCNA TCGGTTCTGACGTGCAAATCGATCGATAGAC-TTGGGTATAGGGGCGAAAGACTAATCGA ** ********************* * Table 2 shows completed identity of species based on sequence comparisons performed with CLUSTAL X and also from GenBank BLAST results. All isolates showed 92 or higher percentage of sequence similarities with existing databases. Most of the isolates identified were diagnoses as S. feltiae. These isolates were mostly from Turkey but also one isolate 5C, from Syria was identified as S. feltiae. Another Syrian isolate, D3, was depicted as most closely related to S. monticolum. However 28S rdna sequences showed 1 base pair difference when compared with the type isolate from South Korea. Another isolate from India, S01, was 29

31 diagnoses as S. abbasi. The Korean isolate 75-2 showed 70% similarity with two Steinernema species: S. hermaphroditum and S. scarabaei. We believe this is a new species that needs to be further characterized. In this respect I have initiated the morphological characterization of this isolate by examining morphological traits of first generation males. Preliminary results of these observations are presented in Table 3. Only one isolate H01 from India was identified as Heterorhabditis indica. This species is widely distributed in this country. 30

32 Table 2.Isolate identity based on results from sequence data Isolate Name Genus Species Country or Region of Origin CK-6 Steinernema S. feltiae Turkey Tur-S3 Steinernema S. feltiae Turkey B02 Steinernema S. feltiae Turkey K4 Steinernema S. feltiae Turkey 60 Steinernema S. feltiae Turkey 5C Steinernema S. feltiae Syria KRK3 Steinernema S. feltiae Turkey S01 Steinernema S. abbasi India D3 Steinernema S. monticolum Syria R03 Steinernema S. feltiae Turkey R04 Steinernema S. feltiae Turkey 75-2 Steinernema Steinernema sp. (new species) South Korea H02 Heterorhabditis H. indica India 2. Phylogenetic analysis Maximum parsimony (MP) analysis of the 28S rdna sequences set yielded 410 parsimony informative characters and produced 166 equally parsimonious trees (Fig. 8) with a tree length of 1153 steps (CI = 0.61). MP analysis placed isolates B02, CK4, KrK3, R03, R04, CK6, Tur-S3 in the S. feltiae clade. This clade comprises Steinernema spp. known to have infective juveniles with small body size (average < 700 μm). Steinernema isolate 75-2 was placed as closely related to S. hermaphroditum and S. scarabaei. Steinernema isolate D3 was placed in a clade that comprises S. monticolum and S. scapterisci. 31

33 ----Summary of PAUP Phylogenetic analysis---- Heuristic search setting: Optimality criterion = parsimony Gaps are treated as missing Multistate taxa interpreted as uncertainty Additional sequence: random Number of replications = 1000 Branch-swapping algorithm: tree-bisection-reconnection (TBR) Number of rearrangement limited to Initial MaxTrees setting = 100 Heuristic search terminated by rearrangement limit (while swapping on tree #1) Total number of rearrangements tried = Score of best tree(s) found = 1153 Number of trees retained = 166 Tree length = 1153 Consistency index (CI) = Homoplasy index (HI) = Retention index (RI) = Rescaled consistency index (RC) = F value = f-ratio =

34 Figure 8. Phylogenetic relationships among Steinernema spp. Single, most parsimonious tree inferred by maximum parsimony analysis of 28S rdna sequences. Numbers represent bootstrap frequencies (1000 replicates). 33

35 Table 3. Patristic distance matrix Below diagonal: Adjusted character distance S.affine 1 S.intermedium 2 6 Steinernema sp Steinernema sp S.feltiae B S.feltiae CK S.feltiae KrK S.feltiae R S.feltiae R S.feltiae CK S.feltiae TurS S.feltiae S.puntauvense S.kraussei S.oregonense S.kushidai S.diaprepesi S.puertoricense Steinernema n. s S.cubanum S.longicaudum S.glaseri S.arenarium S.hermaphroditum S.scarabaei S.khoisanae S.karii S.rarum S.costaricense S.bicornutum S.ceratophorum S.abassi S.abbasi S S.riobrave S.websteri S.anatoliense S.siamkayai S.carpocapsae S.monticolum Steinernema D S.scapterisci C.elegans

36 3. Morphologic analysis Morphological analysis was conducted on isolate 75-2, as it is assumed this isolate represents a new undescribed species. Morphological studies consist on qualitative and quantitative examination of traits of various nematode stages including first and second generation adults (male and female) and third-stage infective juveniles. I was able to examine morphological traits of first generation males. For this purpose, infected G. mellonella cadavers were dissected as indicated in Section 3. Recovery of adult nematode stages, during day 4-5 after initial infection. Males were collected, heat killed and preserved in a small container filled with fixative TAF (triethanolamine and formalin). Nematode morphology was observed in an Olympus BX51 microscope equipped with differential interference contrast (DIC) optics, at various magnifications (10X, 50X, and 100X). A total of 14 morphological characteristics, were measured and calculated. Male morphological characters are the most important in the taxonomy of Steinernematidae. Major morphological features in the anterior portion of the male are: excretory pore location, length of esophagus, position of the nerve ring (Figure 9). Major morphological features of posterior end include: spicule length, gubernaculum length, length and width of tail (at the cloaca level) and position of genital papillae (Table 4). Interestingly enough, the spicule of isolate 75-2 has split-ended, which is a unique morphological feature of this isolate. It well supports the idea of the isolate 75-2 is a new species. 35

37 Table 4. Isolate Morphometric traits of first generation males. specimen TBL MBW AE-EP AE-NR AE-ES TL WA SpL GuL SW GS ratio d ratio e MIN MAX AVG STD TBL=total body length ( from tip of head to end of tail) MBW= width of body taken usually at the midbody level StL= stoma length StW= stoma width NR= distance from anterior end to nerve ring EP distance from anterior end (head) to excretory pore canal opening ES= distance from anterior end (head) to base of esophagus (Basal bulb) WA= width at anus/cloaca (perpendicular to TL) TL= tail length (from anus/cloaca opening to end of tail) ML= mucro length D %= EP divided by ES x 100 E%= EP divided by TL x

38 A A C B B Figure 9A. A: excretory pore opening, B: esophagus length, C: nerve ring position. Figure 9B. A: excretory pore opening, B: anterior portion of esophagus. B B C A A C Figure 10A. A: spicule (yellow), B: gubernaculum (red), C: genital papilla (blue) Figure 10B. A: spicule(yellow), B: gubernaculum(red), C: papilla(blue) 37

39 B A A B Figure 10C. A: spicule, ventral view (red), B: Genital papillae (blue) Figure 10D. A: split-ended spicule, B: gubernaculum showing typical forkshape morphology 38