Nematology 18 (2016) 1015-1043 brill.com/nemy

Molecular and morphological characterisation of americanum-group species (Nematoda: ) from California, USA, and other regions, and co-evolution of bacteria from the genus Candidatus Xiphinematobacter with

Valeria O RLANDO 1,2, John J. CHITAMBAR 3,KeDONG 3,VladimirN.CHIZHOV 4, ∗ Dimitre MOLLOV 5,WimBERT 2 and Sergei A. SUBBOTIN 2,3,4, 1 Via Giacomo Puccini 14, 90017 Santa Flavia, Italy 2 Nematology Research Unit, Department of Biology, Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium 3 Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA 95832-1448, USA 4 Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Leninskii prospect 33, Moscow 117071, Russia 5 National Germplasm Resources Laboratory, USDA, ARS, 10300 Baltimore Avenue, Beltsville, MD 20705, USA Received: 27 May 2016; revised: 26 July 2016 Accepted for publication: 27 July 2016; available online: 15 September 2016

Summary – The -group is a large species complex containing more than 50 species. They are economically important because they are vectors of nepoviruses. The species differentiation of X. americanum-group is problematic because the species share similar morphological characters. In the present study we collected nematode samples from different locations in the USA, Italy and Russia. Six valid species, X. americanum s. str., X. brevicolle, X. californicum, X. pachtaicum, X. rivesi and X. simile, and four unidentified putative Xiphinema species were characterised by morphology and sequencing of D2-D3 of 28S rRNA, ITS1 rRNA and mitochondrial COI genes. New nematode sequences generated totalled 147. Phylogenetic relationships of the X. americanum-group species reconstructed by Bayesian inference for D2-D3 of 28S rRNA gene sequences did not provide clear species delimitation of the samples studied, although the mtDNA presented interspecific variations useful for demarcation among species. Xiphinema americanum s. str., X. californicum, X. pachtaicum, X. rivesi, and two unidentified Xiphinema species were found in 72 soil samples from California. We also reconstructed the phylogenetic relationships using partial 16S rRNA gene sequences within endosymbiotic bacteria of the genus Candidatus Xiphinematobacter and provided solid evidence for distinguishing 17 species of this genus based on the analysis of new and previously published sequences. Fifty-five new bacterial sequences were obtained in the present study and comparison of the bacterial 16S rRNA and nematode COI phylogenies revealed a high level of co-speciation events between host and symbiont. Keywords – 16S rRNA, COI, dagger nematodes, D2-D3 of 28S rRNA, endosymbiotic bacteria, ITS rRNA, virus vector nematodes, Xiphinema americanum s. str., , Xiphinema californicum, Xiphinema pachtaicum, , Xiphinema simile.

The Xiphinema americanum-group is a large species of the X. americanum-group can be distinguished from complex comprising 55 nominal taxa of dagger nema- other Xiphinema by small body length of 1-3 mm, odon- tode (Gutiérrez-Gutiérrez et al., 2012; Archidona-Yuste tostyle 60-200 μm, amphidial fovea stirrup-shaped, didel- et al., 2016). The of the group is controversial phic female reproductive system with genital branches and, as such, species composition depends on the opin- equally developed, uterus short and without differentia- ion of the authority consulted. Lamberti et al. (2000) pro- tion, presence of symbiotic bacteria in the oocytes and posed to define this group based on morphology. Species in the intestines of juveniles and short conoid tail (c <

∗ Corresponding author, e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2016 DOI 10.1163/15685411-00003012 V. Orlando et al.

2.5) with more or less acute terminus, sometimes subdig- X. californicum was questioned by Griesbach & Maggenti itate and uncommon males (Lamberti & Ciancio, 1993; (1990). Lamberti et al., 2000). Several species of the X. america- Hence, the X. americanum-group comprises many spe- num-group are of particular interest because they are vec- cies for which morphological and morphometric charac- tors of Nepovirus (Taylor & Brown, 1997). The species ters overlap, and accurate diagnostics, using the tradi- X. americanum sensu stricto Cobb, 1913, X. bricolense tional approach, remains a rather difficult task. Correct Ebsary, Vrain & Graham, 1989 and X. californicum Lam- identification of these nematodes and a reliable distinction berti & Bleve-Zacheo, 1979 are rated as A1, and X. rivesi between virus vector and non-virus vector species is im- Dalmasso, 1969 as A2, quarantine nematodes by the Eu- perative within a phytosanitary context. Recognising the ropean and Mediterranean Plant Protection Organization complexity of these issues, Lamberti et al. (2002) recom- (EPPO, 2015). mended the use of molecular approaches to discriminate Several species belonging to the X. americanum-group species within the X. americanum-group. have a cosmopolitan distribution. Robbins (1993) and Vrain et al. (1992) and Vrain (1993) were the first to Lamberti et al. (2000) reported 21 species from this group apply PCR-ITS-RFLP for species differentiation and pre- in North America. Three species, X. americanum s. str., X. sented the phylogenetic relationships within the X. ame- californicum and X. rivesi are considered as widespread, ricanum-group. However, it took almost 10 years before seven others are reported to be localised and 11 species researchers finally began to use molecular analysis for are thought to be either rare, or in need of confirmation. characterisation of this group. Ye et al. (2004) sequenced Xiphinema americanum s. str., the type species of the several samples of X. americanum sensu lato and found a genus Xiphinema and natural vector of at least three, pos- minor intraspecific variation in the ITS1 rRNA gene. The sibly four, nepoviruses (Taylor & Brown, 1997), has been authors showed that the X. americanum-group formed a reported particularly in the eastern region of North Amer- separate clade within the genus Xiphinema. Similar re- ica and South Africa, but is absent in the EPPO region sults were obtained by Oliveira et al. (2004), who re- (EPPO, 2015). Xiphinema rivesi, a vector of Cherry rasp ported that four putative species (X. brevicolle, X. dif- leaf virus (CRLV), Tobacco ringspot virus (TRSV) and fusum Lamberti & Bleve-Zacheo, 1979, X. peruvianum Tomato ringspot virus (ToRSV) (Brown et al., 1994) is Lamberti & Bleve-Zacheo, 1979 and X. oxycaudatum considered as the most widespread X. americanum-group Lamberti & Bleve-Zacheo, 1979) of the X. americanum- species in North America (Robbins, 1993). It was origi- group had nearly identical 18S rRNA gene sequences. nally described from France and then reported from sev- However, these species were separated in a clade from eral Central and South American countries (Doucet et al., other Xiphinema species with strong statistical support. 1998) and Europe (Lamberti et al., 2000). Xiphinema cal- Low interspecific variations were also revealed by He ifornicum is a vector of TRSV, ToRSV and CRLV (Brown et al. (2005a) for the D2-D3 expansion regions of 28S et al., 1994). This species is found in several states of rRNA gene sequences of 17 putative species of the X. the USA, Mexico and several countries in South Amer- americanum-group. They showed that X. americanum- ica (Robbins, 1993; Lamberti et al., 2000), but is not group species formed a highly supported clade within present in Europe. Instead, X. pachtaicum (Tulaganov, Xiphinema in the 28S rRNA gene tree. This clade could 1938) Kirjanova, 1951 is widespread in Europe (Siddiqi, be separated into two distinct subclades: X. americanum 1977; Roca et al., 1989; Navas et al., 1990; Decraemer subgroup and X. pachtaicum subgroup (sensu Lamberti & & Robbins, 2007), while it has been reported in Cali- Ciancio, 1993). Further progress in the molecular analysis fornia and Washington, USA, without any morphological of the X. americanum-group was related to the characteri- or molecular confirmation. It has been hypothesised that sation of the mitochondrial cytochrome c oxidase subunit this species was introduced into America from Europe I gene (COI)(Heet al., 2005b; Lazarova et al., 2006; through planting materials (Robbins, 1993). At present, Kumari et al., 2010; Gutiérrez-Gutiérrez et al., 2011), only five species of the X. americanum-group, X. ameri- which provided better species separation and phyloge- canum s. str., X. brevicolle Lordello & Da Costa, 1961, netic resolution, as well as with the characterisation of X. bricolense, X. californicum and X. pachtaicum,have more species and populations (Gozel et al., 2006; Meza been reported in California (Robbins, 1993). The finding et al., 2011; Gutiérrez-Gutiérrez et al., 2012; Zasada et of two of them, X. brevicolle and X. pachtaicum, still re- al., 2014; Archidona-Yuste et al., 2016). Although these quires confirmation and the validity of the identification of works led to a better morphological and molecular de-

1016 Nematology Studies on the Xiphinema americanum-group limitation of some species within the group, several taxa group species from California, USA and other regions; were paraphyletic in the phylogenetic trees, indicating ii) provide a molecular characterisation of populations of that species misidentification and hidden species com- these species using D2-D3 of 28S rRNA, ITS1 of rRNA plexes are likely to have occurred in the published results. and mitochondrial COI sequences; iii) analyse the phy- One of the interesting biological particularities of some logenetic relationships within the X. americanum-group species from the X. americanum-group is the presence species using rRNA and COI gene sequences; iv) esti- of verrucomicrobial endosymbionts (Coomans & Claeys, mate the species boundaries for some Xiphinema using 1998; Coomans et al., 2000; Vandekerckhove et al., 2000, an integrated approach; v) molecularly characterise bac- 2002). Vandekerckhove et al. (2000) described three en- teria from the genus Ca. Xiphinematobacter associated dosymbiont bacterial species, Candidatus Xiphinemato- with Xiphinema; and vi) compare molecular phylogenies bacter americani, Ca. X. rivesi and Ca. X. brevicolli in- of Xiphinema and bacteria and estimate their level of co- side three nematode species: X. americanum s. str., X. speciation. rivesi and X. brevicolle, respectively. It has been shown that these nematode species reproduce by thelytokous Materials and methods parthenogenesis and that the verrucomicrobial endosym- bionts are maternally inherited (Coomans et al., 2000). NEMATODE POPULATIONS Bacteria live as obligate symbionts in the gut epithelium of juveniles, but later they increase in number in the fe- The X. americanum-group species were collected from male ovaries and seem to induce thelytokous parthenogen- cultivated fields and natural areas in different locations esis in their hosts (Coomans & Claeys, 1998; Coomans and host plants in California and a few other states of the et al., 2000; Vandekerckhove et al., 2000, 2002). There USA, in Italy and in Russia (Table 1). In each location, is no evidence for horizontal transfer of these symbionts samples were collected from the upper 40 cm of soil using through the environment, making vertical transmission the a shovel. The soil sample was then kept at 4¡C until the only possible route (Coomans et al., 2000). Indeed, al- nematodes were extracted from soil using a sieving and though the mechanism of this transmission is still un- decanting method (Brown & Boag, 1988) or centrifugal- known, microscopic observations also revealed the pres- flotation (Coolen, 1979). ence of these symbionts in mature oocytes and eggs, thus clearly indicating a transovarial transmission (Coomans et MORPHOLOGICAL STUDY al., 2000). Vandekerckhove et al. (2000) hypothesised a possi- Specimens of some populations were killed and fixed with 4% formalin and 1% glycerin and quickly heated ble long-term co-evolution between X. americanum-group to 70¡C (Seinhorst, 1966). The fixed nematodes were species and their symbionts. It was suggested that a pos- processed to anhydrous glycerin following the glycerin- sible infection of the bacteria into its Xiphinema ances- ethanol method (Seinhorst, 1959) as modified by De Grisse tor occurred during the Jurassic when the X. brevicolle (1969). Light micrographs of nematodes were taken with lineage diverged (Coomans, 1996). Since Ca. X. amer- an automatic Infinity 2 camera attached to a compound icani and Ca. X. rivesi are similar only in 96% of the Olympus BX51 microscope equipped with Nomarski in- 16S rRNA gene sequence, it was supposed that each terference contrast. Measurements were obtained with a Xiphinema species must carry its own specific endosym- camera lucida on a Leitz Wetzlar Ortholux microscope. biotic species (Vandekerckhove et al., 2000). Although, Populations measured and illustrated in this study in- et al. Lazarova (2003, 2004, 2016) revealed a high genetic cluded only those collected in the USA and Italy. diversity of Ca. Xiphinematobacter in Xiphinema,there- lation of these endosymbiotic bacteria with their hosts in DNA EXTRACTION,PCRAND SEQUENCING a phylogenetic framework has not been properly studied. The comprehensive molecular analysis of different popu- Before DNA extraction, a single specimen of dagger lations of X. americanum-group species and their bacteria nematode from each sample was mounted in distilled is still required to test different hypotheses of co-evolution water on a temporary slide in order to take pictures of these organisms. and make measurements of diagnostic characters. The The main goals of the present study were to: i) con- specimen was then cut into four or more pieces with a duct a morphometric characterisation of X. americanum- sterilised needle and transferred to an Eppendorf tube

Vol. 18(9), 2016 1017 V. Orlando et al. 16S rRNA of bacteria Ð KX263198 S.A. Subbotin COI nematode mtDNA of KX263064 KX263203 S.A. Subbotin D2-D3 of 28S rRNA KX263152 KX263151 of nematode Peach Ð KX263051 KX263194 S.A. Subbotin Cherry Ð KX263048 KX263190 S.A. Subbotin Cherry Ð KX263061 KX263189 S.A. Subbotin Walnut Ð KX263046 KX263201 S.A. Subbotin Walnut Ð KX263057 Ð S.A. Subbotin Walnut KX263172 KX263059 Ð K. Dong Walnut KX263174 Ð Ð K. Dong Plum Ð KX263052 Ð J.J. Chitambar Pecan Ð KX263056 Ð J.J. Chitambar Orange KX263171 KX263053 KX263192 S.A. Subbotin Unknown KX263173 KX263054 KX263197 S.A. Subbotin Unknown KX263168 KX263050 KX263196 S.A. Subbotin GrassesUnknown KX263150, Ð KX263058 KX263188 S.A. Subbotin GrapeGrape KX263145, KX263175 KX263062 KX263199 S.A. Subbotin Apricot KX263170 Ð KX263202 S.A. Subbotin Plum KX263169 KX263063 KX263193 S.A. Subbotin used in the present study. X. setariae -group species and Selma County, Westley County, Hughson McFarland McFarland McFarland McFarland Reedley Visalia Avenal Texas Georgia Riverside Alabama County, Ramona Graton Hanford Princeton XA53 USA, California, Fresno County, XA45 USA, California, Stanislaus XA43 USA, California, Stanislaus XA39 USA, California, Kern County, XA37 USA, California, Kern County, XA34 USA, California, Kern County, XA32 USA, California, Kern County, XA25 USA, California, Tulare County, XA24 USA, California, Tulare County, CD1244 USA, California, Kings County, CD1078 Intercepted by CDFA from USA, CD1074 Intercepted by CDFA from USA, CD732CD792 USA, California, Riverside County, Intercepted by CDFA from USA, CD82bCD100 USA, California, San Diego USA, California, Sonoma County, CD70 USA, California, Tulare County, CD48 USA, California, Glenn County, Xiphinema americanum Samples of X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. Table 1. SpeciesX. americanum s. str. Sample code Location Plant host GenBank accession number Collector/Identifier

1018 Nematology Studies on the Xiphinema americanum-group 16S rRNA of bacteria KX263241 V.N. Chizhov Ð Ð S.A. Subbotin COI nematode mtDNA of KX263078 Ð S.A. Subbotin KX263071 Ð S.A. Subbotin KX263107 D2-D3 of 28S rRNA KX263163 KX263144 KX263155 of nematode sp. KX263153 KX263069 Ð S.A. Subbotin PlumCherry KX263162, Ð KX263082 KX263211 S.A. Subbotin Peach KX263165 KX263077 KX263209 S.A. Subbotin Acer Orange KX263156 KX263073 KX263214 S.A. Subbotin Lemon KX263166 KX263067 KX263215 S.A. Subbotin Lemon Ð KX263066 Ð S.A. Subbotin Lemon Ð KX263065 Ð S.A. Subbotin Grape KX263158 Ð KX263217 S.A. Subbotin OrangeOrange KX263143, Orange KX263154, KX263157 Ð Ð S.A. Subbotin Alfalfa Ð KX263060 KX263195 J.J. Chitambar Cherry Ð KX263055 Ð J.J. Chitambar Cherry KX263146 KX263047 Ð S.A. Subbotin Peach Ð Ð KX263191 J.J. Chitambar Peach Ð KX263049 KX263200 S.A. Subbotin Reedley Shafter Reedley Stebbins Cold Canyon Reserve County, Loma Linda County, Goleta Somis Somis County, Ramona Fillmore Fillmore Fillmore Gridley County, Keys County, Patterson County, Patterson Kingsburg XA5XA6 USA, California, Tulare County, USA, California, Kern County, XA4 USA, California, Tulare County, CD1322CD1818 USA, Arizona, Sedona USA, California, Solano County, Tree KX263161 Ð KX263216 S.A. Subbotin CD1247 USA, California, San Bernardino CD231 USA, California, Santa Barbara CD204 USA, California, Ventura County, CD199 USA, California, Ventura County, CD82a USA, California, San Diego CD3CD4 USA,CD5 California, Ventura County, USA, California, Ventura County, USA, California, Ventura County, XA104 USA, California, Butte County, XA93 USA, California, Stanislaus XA70 USA, California, Stanislaus XA61 USA, California, Stanislaus XA54 USA, California, Kings County, CD1816a,b Russia, Stavropol Krai, Kislovodsk Cherry KX263179 KX263106, (Continued.) X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. brevicolle X. americanum s. str. X. americanum s. str. X. americanum s. str. X. americanum s. str. Table 1. SpeciesX. americanum s. str. Sample code Location Plant host GenBank accession number Collector/Identifier

Vol. 18(9), 2016 1019 V. Orlando et al. Ð V. Orlando Ð S.A. Subbotin 16S rRNA of bacteria Ð KX263239 S.A. Subbotin COI nematode mtDNA of KX263113 KX263081 KX263210 S.A. Subbotin KX263116 KX263114, D2-D3 of 28S rRNA KX263139 KX263160 of nematode Unknown KX263138, PlumCherryWalnut KX263164Walnut KX263079 ÐWalnut Ð ÐWalnut KX263072 ÐCherry KX263083 KX263212 S.A. Subbotin Ð KX263159, Cherry S.A. Subbotin KX263205 KX263075Walnut K. Dong KX263207 KX263076 ÐLemon S.A. Subbotin KX263206 ÐPlum S.A. Subbotin KX263080 Ð KX263208Lemon KX263068 Ð KX263182 S.A. Subbotin KX263074Plum KX263111, Ð KX263181 KX263070 Ð KX263109 KX263213 KX263183 J.J. Chitambar S.A. Subbotin KX263110 J.J. Chitambar Ð Ð S.A. Subbotin J.J. Chitambar County Reedley Marysville Wheeler Ridge McFarland McFarland McFarland Marysville Winters County, Goleta Butte City Marysville County, Hughson County, Goleta CD1096CD1101 USA, Minnesota, Saint Paul USA, Minnesota, Saint Paul Grasses Grasses Ð Ð KX263102 KX263103 KX263236 KX263235 D. Mollov D. Mollov V2V3CD787 Italy, Sicily, Palermo, Santa USA, Flavia California, Italy, Mendocino Sicily, Palermo, Santa Flavia Olive Pomegranate Ð Ð KX263115 KX263112 Ð Ð V. Orlando V. Orlando XA8XA14XA36 USA, California, Tulare County, USA, California,XA40 Yuba County, USA, California,XA41 Kern County, USA, California,XA42 Kern County, USA, California, Kern County, USA, California,XA80 Kern County, XA81 USA, California,XA106 Yuba County, USA, California,CD49 Solano Country, USA, California, Santa Barbara USA, California, Glenn County, XA69V1 USA, California, Yuba County, Italy, Sicily, Palermo, Santa Flavia Grape KX263184 KX263108, XA44 USA, California, Stanislaus CD230 USA, California, Santa Barbara (Continued.) X. rivesi X. rivesi X. pachtaicum X. rivesi X. pachtaicum Table 1. Species Sample codeX. californicum Location Plant host GenBank accession number Collector/Identifier X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. californicum X. pachtaicum X. pachtaicum X. pachtaicum X. californicum X. pachtaicum

1020 Nematology Studies on the Xiphinema americanum-group 16S rRNA of bacteria Ð KX263221 S.A. Subbotin COI nematode mtDNA of KX263089 KX263224 S.A. Subbotin KX263091 KX263227 S.A. Subbotin D2-D3 of 28S rRNA KX263129 KX263136 KX263134 KX263176 KX263100 KX263204 D. Mollov of nematode sp. KX263180 KX263117 KX263187 R.V. Khusainov AlfalfaWalnut KX263178Grasses KX263130Grape Ð KX263092 KX263177Pine KX263226 KX263127 KX263229 S.A. Ð Subbotin S.A. Subbotin KX263142 Ð KX263234 C. Ð Blomquist Ð Ð S.A. Subbotin S.A. Subbotin Grape KX263123, ÐTreesRose KX263140Cornus KX263141 KX263104Tomato KX263240 KX263149 ÐPlum S.A. Subbotin KX263119Plum KX263238Plum KX263132 S.A. Ð Subbotin KX263237Grape KX263093 KX263118, S.A. Subbotin Grape KX263135 Ð Ð KX263137 KX263086 KX263090 KX263133, KX263233 S.A. Subbotin S.A. Subbotin S.A. KX263225 Subbotin S.A. Subbotin Grasses and shrubs El Nido Durham Washington Marlatt Park Woodland County, Apple Hill Calistoga County, Moss beach South Carolina region, Sukko Firebaugh Hamilton Orland Butte City Napa Yountville County, San Francisco, Golden Gate Park near the Mississippi bank CD1819CD1841 USA, California, San Mateo CD1815 Intercepted by CDFA from USA, Russia, Krasnodar Krai, Anapa CD1715 USA, California, San Francisco CD1165 USA, Minnesota, Brooklyn Park, sp. 1 CD130sp. 1 CD139sp. 1 USA, California, Merced County, CD863sp. 1 USA, California, Butte County, CD1175sp. 1 USA, Kansas, Manhattan, CD1707 USA, California, Yolo County, USA, California, El Dorado sp. 1 CD96 USA, California, Napa County, sp. 1 CD7sp. 1 CD44sp. 1 CD47 USA, California,sp. Merced 1 County, USA, California, CD50 Glenn County, sp. 1 USA, California, CD91 Glenn County, sp. 1 USA, California, CD95 Glenn County, USA, California, Napa County, USA, California, Napa County, (Continued.) Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema X. rivesi X. rivesi X. simile Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema X. rivesi Table 1. Species Sample codeX. rivesi Location Plant host GenBank accession number Collector/Identifier

Vol. 18(9), 2016 1021 V. Orlando et al. 16S rRNA of bacteria ÐÐ Ð Ð R.V. Khusainov S.A. Subbotin COI nematode mtDNA of KX263085 KX263231 J.J. Chitambar KX263101 Ð J.J. Chitambar D2-D3 of 28S rRNA KX263186 KX263148 KX263131 KX263125 of nematode CherryWalnutWalnut ÐOrange Ð KX263122 KX263087 KX263088 KX263167 KX263084 KX263105Peach Ð KX263230 Ð KX263218 S.A. Subbotin S.A. Subbotin J.J. Chitambar J.J. Chitambar Ð KX263045 Ð K. Dong Plum KX263121, Unknown Ð KX263044 Ð S.A. Subbotin PecanAlfalfaAlfalfa KX263128Alfalfa Ð Ð KX263126Alfalfa KX263097 KX263120Alfalfa KX263098 KX263232 KX263222 KX263099Alfalfa S.A. KX263219 Subbotin J.J. Chitambar Ð KX263228Plum J.J. Chitambar S.A. Subbotin Ð KX263094 Ð KX263124, KX263096 Ð KX263223 KX263095 S.A. Subbotin KX263220 S.A. Subbotin S.A. Subbotin Marysville Winters Winters Calipatria Parlier Marysville Florida Visalia Wheatland Wheatland County, Sacramento County, Elk Grove Dixon Dixon Reedley CD208 Intercepted by CDFA from USA, sp. 1 XA76sp. 1 XA96sp. 1 USA, California, XA100 Yuba County, sp. 2 USA, California, CD152 Solano County, sp. 3 USA, California, Solano County, CD1814sp. 4 USA, California, Imperial County, CA160sp. 5 Russia, Krasnodar Krai, Anapa XA88 USA, Hawaii, Kauai Plum USA, California, Fresno County, KX263185, Ð KX263147, sp. 1 XA65 USA, California, Yuba County, sp. 1 XA7 USA, California, Tulare County, sp. 1 XA12sp. 1 XA13sp. 1 USA, California, XA19 Yuba County, sp. 1 USA, California, XA20 Yuba County, sp. 1 USA, California, XA21 Sacramento sp. 1 USA, California, XA22 Sacramento sp. 1 USA, California, XA26 Solano County, USA, California, Solano County, USA, California, Tulare County, (Continued.) Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema X. setariae Xiphinema Table 1. Species Sample codeXiphinema Location Plant host GenBank accession number Collector/Identifier Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema Xiphinema

1022 Nematology Studies on the Xiphinema americanum-group

Table 2. Primer sets used in the present study. Primer code∗ Sequence (5 → 3) Amplified Reference gene D2A (f) ACA AGT ACC GTG AGG GAA AGT TG D2-D3 of Nunn (1992) 28S rRNA D3B (r) TCG GAA GGA ACC AGC TAC TA rDNA2 TTG ATT ACG TCC CTG CCC TTT Vrain et al. (1992) rDNA5.8S ACG AGC CGA GTG ATC CAC CG ITS rRNA Cherry et al. (1997) COIF (f) GAT TTT TTG GKC ATC CWG ARG COI He et al. (2005b) COIR (r) CWA CAT AAT AAG TAT CAT G XIPHR2 (r) GTA CAT AAT GAA AAT GTG CCAC COI Lazarova et al. (2006) COIF_M13F (f) TGT AAA ACG ACG GCC AGT GAT TTT TTG GKC ATC CWG ARG COI This study COIF_mod_M13F (f) TGT AAA ACG ACG GCC AGT GRT TYT TYG GNC AYT CRG COI This study COIF_He_M13F (f) TGT AAA ACG ACG GCC AGT GAT TCT TTG GAC ACC CGG COI This study 16SF_univ_bact (f) AGA GTT TGA TCC TGG CTC AG 16S rRNA Brosius et al. (1978) Xi_bacter_16SR1 (r) AGC TCY GAG ATT TCA CAC TTG This study M13F (f) (for TGT AAA ACG ACG GCC AGT Ð Messing (1983) sequencing)

∗ (f), forward primer; (r), reverse primer. before adding 2 μl10× PCR buffer and 3 μl proteinase PCR products of 28S rRNA gene were cloned into K (600 μgml−1) (Promega) to a total volume of 20 μl. the pGEM-T Easy vector and transformed into JM109 The tubes were incubated at 65¡C (1 h) and then at 95¡C High Efficiency Cells (Promega). PCR obtained from (15 min). two clones were used for sequencing. Samples were PCR and sequencing were carried out with differ- sequenced by Quintarabio. Newly obtained sequences ent primers depending on the target genes (Table 2). were submitted to the GenBank database under the The partial COI gene were amplified by primer com- accession numbers KX263044-KX263245, which are also binations: COIF or COIF_M13F with XIPHR2; COIF given in Table 1 and in the phylogenetic trees. or COIF_M13F with COIR or using a primer cocktail, COIF_M13F, COIF_mod_M13F, COIF_He_M13F with SEQUENCE AND PHYLOGENETIC ANALYSES one of the reverse primers. The genus-specific Candidatus The newly obtained and published nematode and bacte- Xiphinematobacter primers for amplification of the par- rial sequences for each gene (He et al., 2005b; Lazarova et tial 16S rRNA gene were designed manually based on se- al., 2006, 2016; Kumari et al., 2010; Gutiérrez-Gutiérrez quence differences with closely related genera. et al., 2011, 2012; Sakai et al., 2011; Zasada et al., 2014; PCR mix was prepared as described by Tanha Maafi et Archidona-Yuste et al., 2016, and others) were aligned al. (2003). All PCR reactions were run in a Eppendorf using ClustalX using default parameters (gap opening = 5331 MasterCycler Gradient Thermal Cycler with the 15.00; gap extension = 6.66) for nematode ITS1 and COI following cycling profiles for nematode and bacterial and bacterial 16S rRNA gene sequence alignments and rRNA genes: 4 min at 94¡C, followed by 35 cycles of modified parameters (gap opening = 5.00; gap exten- 1 min at 94¡C, 1 min at 55¡C and 1 min 30 s at 72¡C, sion = 3.00) for the D2-D3 of 28S rRNA gene sequence with a final extension at 72¡C for 10 min; and nematode alignment. The best fit models of DNA evolution were ob- COI gene: 4 min at 94¡C, followed by 40 cycles of 1 min tained for each dataset using the program jModeltest 0.1.1 at 94¡C, 1 min at 45¡C and 1 min 30 s at 72¡C, with a (Posada, 2008) under the Akaike information criterion. final extension at 72¡C for 10 min. PCR products were Bayesian phylogenetic analysis (BI) was carried out using analysed by electrophoresis on agarose gels with ethidium MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). BI anal- bromide. ysis for each gene was initiated with a random starting tree For direct sequencing, the PCR products were purified and was run with four chains for 2.0 × 106 generations. using QIAquick PCR purification kit (Qiagen). Several Two runs were performed for each analysis. The Markov

Vol. 18(9), 2016 1023 V. Orlando et al. chains were sampled at intervals of 100 generations. Af- tion in threshold of 98.65% for differentiating two species ter discarding burn-in samples (10%), a 50% majority rule (Kim et al., 2014). As only partial 16S rRNA gene se- consensus tree was generated. Posterior probabilities (PP) quences were used in our study, the adjustment of this are given on appropriate clades. Sixteen COI sequences percentage was performed as indicated below. were assembled from Illumina libraries for samples de- posited by Zasada et al. (2014) in GenBank and included in the analysis. Pairwise divergences between taxa were Results computed as absolute distance values and as percentage mean distance values based on whole alignment, with ad- Among the original 90 nematode samples included justment for missing data using PAUP∗ 4.0b 10 (Swofford, in the present analysis using a species delimiting ap- 2002). proach integrating morphological taxonomic characters and molecular criteria, we distinguished six species be- CO-PHYLOGENETIC ANALYSES longing to the X. americanum-group including X. ame- ricanum s. str., X. californicum, X. brevicolle, X. pach- Congruence between nematode and bacterial phyloge- taicum, X. rivesi and X. simile Lamberti, Choleva & nies were tested using both global fit and event based Agostinelli, 1983, and also five unidentified putative methods. Procrustean Approach to Cophylogeny (PACo) Xiphinema species (Table 1). Xiphinema setariae from (Balbuena et al., 2013) in R using packages ape and ve- one Floridian sample was also included in this study. In gan was applied to evaluate and test the global fit be- 72 X. americanum-group samples collected from 25 coun- tween the nematode and bacterial trees. The significance ties of California, X. americanum s. str. was found in 20 of the statistic was determined using a permutation test samples, X. californicum in 22 samples, X. pachtaicum in with 100 000 runs. Jane 4 (Conow et al., 2010) with event- three samples, X. rivesi in three samples, and Xiphinema based reconstruction method was used with the follow- sp. 1 in 23 samples and Xiphinema sp. 2 and Xiphinema ing default event-cost scheme (co-speciation = 0, duplica- sp. 5 each occurred in one sample. A mixture of two tion = 1, host switch = 2, sorting = 1, failure to diverge = species, namely X. americanum s. str. and X. californicum, 1), a number of generations of 100, population size of was detected for one soil sample. Xiphinema rivesi was 100 and sample size of 50. The probability of observing found in California from natural areas only, whereas other the inferred number of potential co-speciation events by species were collected from orchards. chance was calculated by random tip mapping. ‘Prevent mid-polytomy’ option was applied to ensure that no co- MORPHOLOGICAL AND MORPHOMETRIC evolutionary event was involved in the branches created CHARACTERISATION OF Xiphinema SPECIES to resolve polytomies. Populations of five species of the X. americanum-group NEMATODE AND BACTERIA SPECIES IDENTIFICATION were identified on the basis of morphology (Figs 1-4) AND DELIMITATION and morphometrics (Tables 3-4). Due to an overlap of measurements, mean morphometric values were primar- Nematode specimens from each location were identi- ily used to differentiate the species with the aid of a poly- fied by light microscopy using morphological and mor- tomous key and compared with the original and the few phometric characters according to the polytomous keys subsequent descriptions. Fixed female specimens of these published by Lamberti et al. (2000, 2004) and species de- populations had the general characteristics of X. ameri- scriptions (Lamberti & Bleve-Zacheo, 1979; Lamberti & canum s. l., including body C-shaped when specimens Golden, 1984). Species delimitation of Xiphinema in this are killed and fixed, gradually narrowing at both extremi- study was performed using an integrated approach based ties, vulva at mid-body and reproductive system didelphic on morphological and morphometric analyses combined with equally developed branches. Males were not found. with molecular based phylogenetic inference (tree-based Brief descriptions of distinguishing morphological char- methods) and sequence analyses (genetic distance meth- acters used in the identification for each species are pro- ods) (Sites & Marshall, 2004). vided here. Species delimitation of Ca. Xiphinematobacter was Xiphinema pachtaicum (Table 3; Figs 1A-D; 2A-D) made based on the results of phylogenetic inference and was characterised by a lip region distinctly offset by sequence analyses of 16S rRNA gene with a recommenda- a deep constriction, anteriorly flattened. Robust odon-

1024 Nematology Studies on the Xiphinema americanum-group

Fig. 1. Photomicrographs of anterior region of females. A-D: Xiphinema pachtaicum (CD49, V1, V2a, V2c); E, F: X. americanum s. str. (CD48, CD1074); G, H: Xiphinema sp. 1 (CD96, CD50). (Scale bar = 10 μm.)

Vol. 18(9), 2016 1025 V. Orlando et al.

Fig. 2. Photomicrographs of posterior region of females. A-D: Xiphinema pachtaicum (V1, V2a, CD49, CD49); E-H: X. americanum s. str. (CD48, XA39, CD1244, XA34); I-L: Xiphinema sp. 1 (CD47, CD50, CD91, XA20). (Scale bar = 10 μm.)

1026 Nematology Studies on the Xiphinema americanum-group

Fig. 3. Photomicrographs of anterior region of females. A: Xiphinema californicum (XA44); B: X. rivesi (CD1715); C: X. americanum s. str. (CD82b); D: X. rivesi (CD1101). (Scale bar = 10 μm.) tostyle. Vulva at 48-57% of the total body. Tail conoid ventrally straight with slight pointed tip and a large anal with a narrowly rounded tip. body diam. Xiphinema americanum s. str. (Table 3; Figs 1E, F; 2E- For Xiphinema sp.2(Fig.4G),Xiphinema sp. 3, H; 3C; 4H) was characterised by a rounded lip region Xiphinema sp. 4 and Xiphinema sp. 5, no morphometric offset by a slight constriction. Vulva at 42-65% of the data are available in the current study. Further studies are body. Long tail, dorsally curved with slightly pointed tip. needed to define and comprehensively describe these pu- Xiphinema californicum (Table 4; Figs 3A; 4A-D) was tative species that are herein identified only molecularly. characterised by a rounded lip region, anteriorly flat and distinctly offset from the body. Long odontostyle. Vulva at MOLECULAR CHARACTERISATION OF Xiphinema about mid-body. Tail conoid, dorsally curved with slightly SPECIES pointed tip. Xiphinema rivesi (Table 4; Figs 3B, D; 4E, F, I-L) The D2-D3 of 28S rRNA gene was characterised by a large lip region (11.0-12.5 μm) The D2-D3 alignment was 816 bp long and included anteriorly flat and almost continuous with the body. Vulva 179 X. americanum-group sequences, and two outgroup near middle of body. Tail long, conoid, ventrally straight sequences (X. diversicaudatum and X. index). Sixty- with rounded to slightly narrowly rounded tip. nine new gene sequences of the D2-D3 domain of 28S Xiphinema sp. 1 (Table 4; Figs 1G, H; 2I-L) was rRNA were obtained in the present study. Sequence vari- characterised by a large body length, more or less C- ation (uncorrected p-distance) for Clade I reached 3.4% shaped body after fixation. Lip region rounded, slightly (19 bp), whereas for Clade II the value was 17.5% expanded, anteriorly flat, almost continuous with body. (106 bp). Instraspecific variation for X. pachtaicum was Vulva position at 42-54% of total body. Tail conoid, 0.0-0.75% (0-7 bp). The BI tree contained two highly sup-

Vol. 18(9), 2016 1027 V. Orlando et al.

Fig. 4. Photomicrographs of posterior region of females. A-D: Xiphinema californicum (CD1247, XA41, XA44, CD3); E, F: X. rivesi (CD1715); G: Xiphinema sp. 2 (CD152); H: X. americanum s. str. (CD82b); I-L: X. rivesi (CD1165, CD1101, CD1101, CD1096). (Scale bar = 10 μm.)

1028 Nematology Studies on the Xiphinema americanum-group 1 2 64 11 6 7 3 2 164 4 3 5 1.4 0.7 0.35 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 437 570 548 60 909 15 268 651 941 585 120 126 1898 658 433 0.6 7.1 1.2 9.5 0.22 1.9 ± ± ± ± ± ± ± ± ± ± ± ± ± s.d. (range). ± ± ± 76051 97 1683 1762 42835 67365 35052 64 840 907 25655 25 280 282 48487 11718 55554 0.2 1.6 1.9 23133 0.666.3 ± ± ± ± ± ± ± ± ± ± ± ± ± m and in the form: mean ± μ 434 163 348 9 52.3 243 30 282 782 959 240 1772 130 166 848 0.9 6.3 0.2 1.74 ± ± ± ± ± ± ± ± ± ± ± ± ± 67 955 435 350 855 643 13 281 12020 786 459 112 1914 332 11 71 0.2 1.8 1.05 8.4 8.4 8.4 11 0.35 7.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± females. All measurements are in 334 373 253 130 1050 644 51 272 220 387 765 214 1994 0.8 8.8 10 59 0.6 7.2 0.2 1.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± X. americanum s. str. and 129 276 254 42 1001 163 648 38 284 118 283 159 13131 105 1833 0.7 9.1 X. pachtaicum X. americanum s. str. 1.03 6.5 0.04 1.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 52 1849 129 373 153 28 979 364 550 40 303 217 484 164 129 0.6 8.7 1.3 6.2 0.2 1.6 ± Xiphinema pachtaicum ± ± ± ± ± ± ± ± ± ± ± ± ± ± V1 V2 V3 CD49 CD48 CD70 XA32 XA34 XA39 (6-9) (5-7) (6-7) (7.0-7.5) (6-8) (6-8) (5-7) (6-8) (8-10) (8.4-9.8) (8.4-9.8) (8.4-11) (8.4-11) (8.4-11) (49-52) (51-55) (52-57) (48-56) (46-53) (42-52) (45-65) (43-50) (51-58) (63-65) (50-72) (46-68) (43-69) (38-65.5) (42-60) (45-62) (45-56) (44-56) (44-56) (35-53) (39-45) (42-47) (45-70) (33-56) (78-87) (81-86) (78-86) (78-98) (78-98) (75-87) (84-98) (81-89) (19-22) (17-20) (17-19) (17-21) (17-21) (17-20) (17-21) (56-59) (63-65) (51-65) (59-72) (51-75) (51-65) (43.6-61) (50.8-60) (28-30) (28-30) (28-35) (31-33) (28-34) (28-42) (28-36) (30-33) (28-30) (28-33) (31-42) (31-42) (28-42) (28-42) (31-45) (1.3-2.0) (1.5-1.6) (1.4-2.0) (1.4-2.0) (1.5-2.1) (1.4-2.0) (1.6-2.1) (1.4-2.6) 1.7 (196-302) (269-350) (246-358) (252-285) (252-314) (244-322) (252-296) (252-280) (1654-1792) (1764-2002) (1764-2002) (1820-2128) (1724-2312) (1596-1890) (1526-1954) (1708-2184) Morphometrics of guiding ring (72-81) (70-75) (73-78) (59-84) (70-73) (56-70) (56-70) (67-73) to vulva (834-901) (935-1036) (854-1148) (980-1148) (812-1232) (747-938) (834-994) (817-1008)  c Tail length 31 Anterior end to 75 V51 irgoda.8.7 Lipregiondiam. c55 Anterior end 879 Odontophore 48 Pharynx 260 b6.8 Odontostyle 85 Anal body diam. 19 a57 Table 3. Character nL 54475101910 1734 Body diam. 30

Vol. 18(9), 2016 1029 V. Orlando et al. 84.8 57.2 4.9 42.4 2.4 6.5 4.0 2.1 0.5 1.1 25.8 14.1 0.8 12.3 0.07 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (11-14) 86 1322.6 20 347.5 1 31.5 4 32.2 3 48.9 6 90.2 121 2425.2 84 76.2 54.4 24 105.8 68.7 4 49.6 0.4 7.9 0.1 1.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± s.d. (range). ± m and in the form: mean 19 2058, 2254 2035 μ 47 1092, 1092 1083 24 294, 322 303 22530 2 31, 36 35 1 34, 36 45 67070 5 62, 67 59 2 48, 53 53 414752 87, 90 88 3 61, 62 45 0.576.7 0.16 1.2, 1.4 1.1 X. californicum X. rivesi ± ± ± ± ± ± ± ± ± ± ± ± ± ± females. All measurements are in X. rivesi sp. 1 and Xiphinema X. californicum 94 896, 924 1036, 1274 1162, 1176 999 96 1848, 1890 2142, 2385 2156, 2198 2033 16 252, 266 274, 288 308, 322 314 1 21 19 21, 28 22 2 31, 35 35 35 29 6 31, 42 36, 42 28, 36 31 9 59, 62 76, 84 84 75 5 45, 49 48, 53 53, 54 49 6 53, 61 61, 68 61, 63 70 11 44, 61 51, 65 60, 77 65 0.4 6.9, 7.5 7.8, 8.3 6.8, 7 6.5 0.3 1.4, 2 1.8, 2.1 1.3 1.4 1.9 56 42 48, 56 46 2.8 84, 87 87, 92 87, 92 93 ± ± sp. 1, ± ± ± ± ± ± ± ± ± ± ± ± (17-21) (21-25) (28-31) (29-35) (28-36) (28-31) (42-47.6) (44-55) (28-45) (28-33) (28-42) (27-37) (42-54) (46.4-51) (47-61) (52-57) (42-47) (45-48) (45-56) (52-86) (45-73) (60-72) (49-77) (34.8-96.4) (84-91) (89-98) (84-90) (99-113) (56-74) (66-72) (41-51) (46.6-55.3) (6.6-8.1) (6-7.3) (5.8-7.2) (6.1-11.7) (1.4-2.5) (1.2-1.6) (0.9-1.3) (0.9-1.1) (280-322) (283-336) (274-339) (290-383) (1193-2268) (2007-2058) (1795-2198) (2250-2595) Xiphinema 1.8 1.9 CD44 CD47 CD50 CD139 XA19 CD3 XA42 CD1101 CD1819 Morphometrics of to vulva (901-1162) (938-1050) (969-1288) (1233-1461) guiding ring (45-70) (70-84) (59-78) (59-98)  Anterior end 1036 1003 Pharynx 322 292 Anal body diam. 20 20 Body diam. 31 32 Tail length 35 39 c Anterior end to 84 58 Lip region diam. 8.4 11 8.4, 11 8.4 11 8.4 8.4 11 12.5 c6055 V4948 Odontophore 45 44 b6.67.2 Odontostyle 87 88 a6966 Table 4. Character nL 1822252116 2114 2088

1030 Nematology Studies on the Xiphinema americanum-group

Fig. 5. Phylogenetic relationships within the Xiphinema americanum-group. Bayesian 50% majority rule consensus tree as inferred from the analysis of the D2 and D3 expansion segments of 28S rRNA sequence alignment under a GTR + I + G model. Newly obtained sequences are indicated in bold.

Vol. 18(9), 2016 1031 V. Orlando et al.

Fig. 5. (Continued.) ported clades (Fig. 5). Clade I (PP = 100%) included 0-8.7% (0-32 bp), X. pachtaicum 0-2.7% (0-10 bp), X. X. americanum s. str., X. californicum, X. rivesi, X. bre- simile 0-6.6% (0-21 bp), Xiphinema sp. 1 0-13.3% (0- vicolle and several other species. Relationships between 48 bp), X. rivesi 0-15.6% (0-56 bp), X. brevicolle species species of this clade were not well resolved. Clade II complex 0-21.4% (0-75 bp). In the COI treeshowninFig- (PP = 99%) consisted of X. pachtaicum, X. incertum ure 7 the samples from the X. brevicolle complex were Lamberti, Choleva & Agostinelli, 1983, X. parapachyder- distributed across six subclades (A-F), the first subclade mum Gutiérrez-Gutiérrez, Cantalapiedra-Navarrete, De- group (A-D subclades, PP = 97%) included isolates iden- craemer, Vovlas, Prior, Palomares-Rius & Castillo, 2012, tified by other researcher as X. brevicolle s. str., X. dif- X. pachydermum Sturhan, 1983, X. simile and several fusum and X. taylori Lamberti, Molinari, Moens & Brown, other species. Relationships between the majority of 1992 with intraspecific divergences up to 15.3% and the species in this clade were well resolved. second subclade group (F and E subclades) comprised X. Diagnostics of species of the X. americanum-group brevicolle s. str. from the type locality and X. incognitum belonging to Clade I cannot be reliably differentiated Lamberti & Bleve-Zacheo, 1979 from China, respectively, using a tree building method and a signature sequence with intraspecific divergences up to 19.7%. Interspecific approach is proposed to make identification. The 100 bp divergence varied from 13-17% for X. americanum s. str. fragment of the D2 segment of 28S rRNA gene sequence and X. californicum and from 13-18% for X. rivesi and is proposed here as a signature sequence for identification Xiphinema sp. 1. of some X. americanum-group species (Fig. 6). Phylogenetic relationships within all species are given in Figure 7. Five major clades (I-V) are indicated in The mitochondrial COI gene this tree. Several subclades or groups marked by capital The mitochondrial COI gene alignment included 155 letters (A-F) were distinguished within X. californicum, sequences of the X. americanum-group and two se- Xiphinema sp. 1, X. americanum s. str.,theX. brevicolle quences, X. diversicaudatum and X. index selected as out- complex, and X. simile. group taxa. The alignment length was 373 bp. A total of 74 new COI gene sequences were obtained in the present The ITS1 rRNA gene study. Intraspecific variations for the studied species were: The ITS1 rRNA gene alignment contained 14 se- X. americanum s. str. 0-10.0% (0-35 bp), X. californicum quences of the X. americanum-group and two outgroup

1032 Nematology Studies on the Xiphinema americanum-group

Fig. 6. Fragment of the D2 segment of 28S rRNA gene alignment with signature sequences for some Xiphinema americanum-group species. Numbers in brackets after species name indicates other putatively identified species under similar sequence fragment: (1) X. diffusum;(2)X. rivesi and X. thornei;(3)X. californicum and X. pacificum;(4)X. citricolum;(5)X. tarjanense. Numbers in brackets after accession number is the number of sequences.

Vol. 18(9), 2016 1033 V. Orlando et al.

Fig. 7. Phylogenetic relationships within the Xiphinema americanum-group. Bayesian 50% majority rule consensus tree as inferred from the analysis of the partial COI sequence alignment under a HKY + I + G model. Posterior probabilities more than 70% are given for appropriate clades. New sequences obtained in this study are in bold. Identification of some species made in this study are given in the right from vertical bars. Original species identification are provided in appropriate clades.

1034 Nematology Studies on the Xiphinema americanum-group

Fig. 8. Phylogenetic relationships within species of the clade II of the Xiphinema americanum-group as defined by Archidona-Yuste et al. (2016). Bayesian 50% majority rule consensus tree as inferred from the analysis of the ITS1 rRNA sequence alignment under a GTR + I + G model. Newly obtained sequences are indicated in bold. sequences and was 1088 bp in length. Four new sequences genetic distance between them (r = 0.948). Calculated of two Xiphinema species were obtained in the present threshold for the partial sequence similarity was estimated study. Phylogenetic relationships within species of Clade in our study as 97.7%, which was used for bacterial II of the X. americanum-group, as defined by Archidona- species delimiting. In the results of our analysis we Yuste et al. (2016), are presented in Figure 8. distinguished 17 species of Ca. Xiphinematobacter: Ca. X. rivesi, Ca. X. americani, Ca.X.brevicolleand 14 MOLECULAR CHARACTERISATION OF Candidatus unidentified species of Ca. Xiphinematobacter.Fiveof Xiphinematobacter SPECIES these 14 unidentified species are reported for the first time.

Species delimiting for bacteria Phylogenetic relationships Kim et al. (2014) proposed a threshold of 98.65% 16S rRNA gene sequence similarity for species demarcation The partial 16S rRNA gene sequence alignment in- of bacterial species. This threshold was calculated for the cluded 81 Ca. Xiphinematobacter and two outgroup se- complete length of the 16S rRNA gene. In our study, quences and was 575 bp in length. Fifty-five new bac- we only partially sequenced this gene in our samples. terial sequences were obtained in the present study. The The comparison of published full length sequences with phylogenetic relations between species and isolates of this their partial length revealed a high level of correlation in genus are given in Figure 9. The BI tree contained four

Vol. 18(9), 2016 1035 V. Orlando et al.

Fig. 9. Phylogenetic relationships within Candidatus Xiphinematobacter. Bayesian 50% majority rule consensus trees as inferred from 16S rRNA gene sequences alignment under a GTR + I + G model. Newly obtained sequences are indicated in bold.

1036 Nematology Studies on the Xiphinema americanum-group

Fig. 10. Co-phylogenetic relationships between the endosymbionts Candidatus Xiphinematobacter (right, 16S rRNA tree) and their hosts Xiphinema americanum-group species (left, COI tree). Bayesian 50% majority consensus trees with posterior probability values more than 70% for appropriate clades. Dotted lines show the association between a X. americanum-group species and its symbiont. major clades. Relationships between these clades were 10 for reconstructions (Fig. 11). The p-value of Jane anal- unresolved. ysis was less than 0.01 and, thus, significant co-speciation events were found in the results of analysis. All individual CO-PHYLOGENETIC ANALYSIS nematode-bacteria associations were statistically signifi- cant (Fig. 12). Each nematode species corresponds with a unique species or a group of species of Ca. Xiphinematobacter. Species of the X. brevicolle complex carry four species Discussion (Ca. X. brevicolli, Ca. Xiphinematobacter sp. 2, Ca. Xiphinematobacter sp. 10 and Ca. Xiphinematobacter sp. NEMATODE SPECIES DELIMITATION AND 11), X. rivesi three species (Ca.X.rivesi, Ca. Xiphine- IDENTIFICATION matobacter sp. 6 and Ca. Xiphinematobacter sp. 7), X. californicum two species (Ca. Xiphinematobacter sp. 4 In the present study we applied an integrated ap- and Ca. Xiphinematobacter sp. 9) and all other Xiphinema proach combining morphological and morphometric anal- species have only one species (Fig. 9). yses with phylogenetic inference and sequence analyses For the co-phylogenetic study, 11 species of Xiphinema of the D2-D3 of 28S rRNA and partial COI genes for and 15 bacterial species were included in the reduced species delimitation of X. americanum-group species. The dataset. The BI trees with nematodes (COI tree) and as- remarkable complications in this procedure are that the sociated bacteria (16S rRNA tree) are given in Figure 10. analysis of the partial 28S rRNA gene sequences fails The global test with PACo indicated a significant con- in species differentiation of some Xiphinema species and gruence between nematode and bacterial trees (m2 global that interspecific and intraspecific divergences in COI for value = 0.15143, p<0.0001). The event-based anal- these nematodes are higher than for other organisms. For ysis with Jane, taking tree topologies into account, also example, intraspecific nucleotide sequence variation for yielded a highly significant congruence between host and helveticus Lamberti, Kunz, Grunder, Moli- symbiont trees. The co-phylogenetic analysis with Jane nari, De Luca, Agostinelli & Radicci, 2001 reached 7.3% yielded 291 scenarios. The number of events in these sce- (Kumari & Subbotin, 2012) and 9.5% for X. diversicauda- narios were: co-speciations = 16 or 17, duplication and tum (Subbotin et al., unpubl.) and 15.5% for L. orientalis host switches = 4 or 5, losses = 0 or 2 with total cost = Loof, 1983 (Subbotin et al., 2015).

Vol. 18(9), 2016 1037 V. Orlando et al.

Fig. 11. Reconciliation between Candidatus Xiphinematobacter (16S rRNA tree) and Xiphinema (COI tree) phylogenies. One of 291 isomorphic solutions with 17 co-speciations, four duplications and host switches, and two losses (total cost = 10). The reconciliation of bacteria and host trees was generated with Jane 4. Thin and thick lines represent bacteria and their nematode hosts, respectively. Empty circles represent co-speciations, arrows represent host switches, and dashed lines represent sorting events.

Fig. 12. Contribution of each Xiphinema Ð Candidatus Xiphinematobacter association to a general co-evolution. Each bar represents a jack-knifed squared residual and error bars represent upper 95% confidence intervals. The dashed line indicates the median squared residual value.

1038 Nematology Studies on the Xiphinema americanum-group

Xiphinema americanum s. str. and X. californicum grapevine and peach trees (Robbins & Brown, 1991; Several nematode samples obtained in this study were Robbins, 1993). Our finding of X. rivesi in California is identified, based on morphological characters and mor- the first record of this species for this state. Lamberti & phometrics, as representatives of X. californicum. Stud- Bleve-Zacheo (1979) reported that American (Kansas and ied Californian populations were in agreement with mor- Nebraska) populations of X. rivesi differed from the type phological descriptions and morphometric values for the population (France) by a shorter odontostyle (80-90 vs type population of X. californicum reported by Lamberti 91-101 μm). In our study the shorter stylet lengths were & Bleve-Zacheo (1979). Although Griesbach & Maggenti found in the Minnesota population (84-90 μm), whereas (1990) synonymised X. californicum with X. americanum longer stylet lengths were observed in the Californian μ s. str. on the basis of a morphometric analysis of speci- population (99-113 m). mens from Pennsylvania, New York and California, Cho Xiphinema pachtaicum & Robbins (1991) used a similar approach and came to This species was previously recorded from California the conclusion that these species were different from each and Washington (Robbins, 1993) without any morpholo- other and subsequently did not support the synonymy pro- gical and molecular confirmation. It has been suggested posed by Griesbach & Maggenti (1990). In the current that this species was most probably introduced from Eu- study, we recognise X. californicum as a valid taxon, al- rope with plant materials. In our present study, we con- though it still requires additional detailed morphological firmed the presence of X. pachtaicum in the USA by and molecular delimiting using topotype material. We morphological and molecular studies for the first time. should note that the signature sequence of the D2 seg- The location of the vulva in Californian populations of ment of an X. californicum sample identified by He et X. pachtaicum was slightly more anterior, and therefore al. (2005a) is identical to those of X. americanum s. str. lower in value, than reported for the holotype specimen by and X. pacificum and differs from those of X. californicum Lamberti & Siddiqi (1977), and subsequently observed in obtained in this study. It should be also noticed that all various global populations by Lamberti & Bleve-Zacheo Californian populations of X. americanum s. str. were in (1979). Similarly, tail length was slightly longer for one agreement with the morphological descriptions and mor- Californian population (CD49) than for the other popula- phometric mean values for this species as reported by tions, as well as those reported by the previously refer- Lamberti et al. (2000) and Lamberti & Golden (1984). enced scientists and Lamberti et al. (2000). Xiphinema brevicolle s. l. We should note that in our original dataset no species were characterised from their type localities, and there- In our study, one isolate from Russia was identified fore diagnostics of our samples have been made based on as a representative of the X. brevicolle complex. After morphology or/and similarity with previously identified studying type specimens and following a critical review and published sequences. The characterisation and identi- of the article of Lamberti et al. (1992) with descriptions fication of these species still require additional verification of X. brevicolle s. str., X. diffusum, and several other and molecular comparisons with the type material. We are species, Luc et al. (1998) concluded that X. diffusum, aware that future molecular analyses of type material of X. incognitum, X. parvum Lamberti, Molinari, Moens the studied species may contradict some of the results ob- & Brown, 1992, X. pseudoguirani Lamberti, Molinari, tained in this work. Moens & Brown, 1992, X. sheri Lamberti & Bleve- Zacheo, 1979 and X. taylori should be considered as Candidatus Xiphinematobacter DIVERSITY IN junior synonyms of X. brevicolle. However, our molecular NEMATODES study showed that the X. brevicolle complex had a higher value of intraspecific variation suggesting the presence of Our study confirms the conclusions made by Lazarova several cryptic species within these samples. et al. (2004, 2016) that the partial 16S rRNA gene se- quence analysis revealed a high diversity of endosymbi- Xiphinema rivesi otic bacteria isolated from samples of X. americanum- It had been thought that X. rivesi was distributed mainly group. After the analysis of 37 sequences, Lazarova et al. in the eastern States only, where it has been identified as (2004) distinguished three main groups: i) pachtaicum- a vector of tomato ringspot nepovirus and associated with simile; ii) brevicolle-diffusum-taylori; and iii) mainly the spread and transmission of viruses to apple, cherry, americanum s. str. within studied bacteria and 11 species,

Vol. 18(9), 2016 1039 V. Orlando et al. eight of them being considered as potentially new species. transmission of bacteria from one nematodes species In a recent study, Lazarova et al. (2016) differentiated to another seems to be most unlikely as only a few nine phylotypes within studied bacteria, six phylotypes host switching events were observed in the reconciliation not previously having been reported. reconstructions. In our study, species delimiting with the partial 16S Recently, comparative analysis of the complete genome rRNA gene allowed us to distinguish 17 species of of Ca. Xiphinematobacter with other Verrucomicrobia Ca. Xiphinematobacter, 14 of them being putative new revealed extreme genome reduction, massive losses of species. The bacterial species demarcated in the cur- genes, including those responsible for locomotion and rent study correspond with the phylotypes proposed by chemotaxis, and gene set enrichment for several biolog- Lazarova et al. (2016) in the following way: Ca. Xiphine- ical functions consistent with the hypothesis that this en- matobacter sp. 3 = Phylotype A; Ca. Xiphinematobac- dosymbiont plays the role of a nutritional mutualist for the ter sp. 5 = Phylotype B; Ca. Xiphinematobacter sp. 10 host (Brown et al., 2015). It has been also suggested that and Ca. Xiphinematobacter sp. 11 = Phylotype C; Ca. such features are characteristic of endosymbiotic bacteria Xiphinematobacter sp. 12 = Phylotype D; Ca.X.brevi- with long-lasting relationships with their hosts, an infer- colli and Ca. Xiphinematobacter sp. 2 = Phylotype E; Ca. ence consistent with our results that showed a strong co- Xiphinematobacter sp. 13 = Phylotype F; Ca. Xiphine- evolution pattern with the hosts. matobacter sp. 14 = Phylotype G; Ca. X. rivesi and Ca. Thus, in this study the patterns of co-divergence of Xiphinematobacter sp.1 = Phylotype H; Ca. X. ameri- X. americanum-group with endosymbiotic bacteria are cani = Phylotype I. proved for the first time. An accurate study of the Several attempts to amplify a partial 16S rRNA gene endosymbiotic bacteria could, in future, be useful in using a species-specific primer from certain nematode facilitating robust co-evolutionary studies and to discover samples have failed, an outcome which may indicate that novel associations between nematode and bacteria. this bacterium is in low number or absent in these ne- matode isolates. Considering that the symbiotic bacte- ria of most currently recognised X. americanum-group Acknowledgements species have not yet been described, future research using transmission electron microscopy, scanning electron mi- Valeria Orlando thanks for a financial support from Si- croscopy, and fluorescent in situ hybridisation are needed ciliafuturo, MISURA 4, Programma Operativo del Fondo to characterise the novel candidate verrucomicrobial bac- Sociale Europeo dell’ Obiettivo Convergenza della Re- teria, Ca. Xiphinematobacter in each host. gione Siciliana. Drs S.A. Subbotin and V.N. Chizhov acknowledge support from the Russian Foundation of CO-EVOLUTION OF BACTERIA WITH NEMATODES Basic Research, project number 14-04-00953, and Dr W. Bert acknowledges support from the special research Our analysis revealed a high level of specificity for fund UGent 01N02312. Drs J. Chitambar and S.A. Sub- host-endosymbiont relationships between nematodes and botin acknowledge support from the United States De- bacteria and showed that each nematode species carries partment of Agriculture and Plant Health In- its own unique bacterial species. In several cases, two spection Service, National Cooperative Agricultural Pest or more bacterial species were found for one nematode Survey (USDA-APHIS-CAPS) program for 2005-2008, species. The occurrence of high specificity bacteria asso- 2012. The authors thank Drs R. Inserra and W. Decrae- ciation could have an important application in nematode mer for corrections and suggestions during the manuscript diagnostics, where identification of Xiphinema species preparation and Drs C. Blomquist, J. Stack and R.V. Khu- could be done just by using 16S rRNA gene sequence in- sainov for providing soil samples and nematode materi- formation of the associated bacteria. als. In order to understand the evolutionary pattern of bacteria in nematodes, we used both new and published sequences of genetic markers from hosts and symbionts References to determine co-phylogenetic patterns on a global scale. Our results indicate patterns of co-evolution and showed Archidona-Yuste, A., Navas-Cortes, J.A., Cantalapiedra- that bacteria exhibited strong co-speciation with their Navarrete, C., Palomares-Rius, J.E. & Castillo, P. (2016). nematode host. The role of environmental horizontal Cryptic diversity and species delimitation in the Xiphinema

1040 Nematology Studies on the Xiphinema americanum-group

americanum-group complex (Nematoda: ) Doucet, M.E., Ferraz, L.C.C.B., Magunacelaya, J.C. & Brown, as inferred from morphometrics and molecular markers. D.J.F. (1998). The occurrence and distribution of Longidori- Zoological Journal of the Linnean Society 176, 231-265. dae (Nematoda) in Latin America. Russian Journal of Nema- Balbuena, J.A., Mõguez-Lozano, R. & Blasco-Costa, I. (2013). tology 6, 111-128. PACo: a novel procrustes application to cophylogenetic anal- EPPO (2015). EPPO Standards. EPPO A1 and A2 lists of ysis. PLoS ONE 8, e61048. pests recommended for regulation as quarantine pests. Avail- Brosius, J., Palmer, M.L., Kennedy, P.J. & Noller, H.F. (1978). able online at http://archives.eppo.int/EPPOStandards/PM1_ Complete nucleotide sequence of a 16S ribosomal RNA gene GENERAL/pm1-02(24)_A1A2_2015.pdf. from Escherichia coli. Proceedings of the National Academy Gozel, U., Lamberti, F., Duncan, L., Agostinelli, A., Rosso, of Sciences of the United States of America 75, 4801-4805. L., Nguyen, K. & Adams, B.J. (2006). Molecular and mor- Brown, A.M.V., Howe, D.K., Wasala, S.K., Peetz, A.B., Zasada, phological consilience in the characterization and delimita- I.A. & Denver, D.R. (2015). Comparative genomics of a tion of five nematode species from Florida belonging to the plant-parasitic nematode endosymbiont suggest a role in Xiphinema americanum-group. Nematology 8, 521-532. nutritional symbiosis. Genome Biology and Evolution 7, Griesbach, J.A. & Maggenti, A.R. (1990). The morphometrics 2727-2746. of Xiphinema americanum sensu lato in California. Revue de Brown, D.J.F. & Boag, B. (1988). An examination of methods Nématologie 13, 93-103. used to extract virus-vector nematodes (Nematoda: Longi- Gutiérrez-Gutiérrez, C., Castillo, P., Cantalapiedra-Navarrete, doridae and Trichodoridae) from soil samples. Nematologia C., Landa, B.B., Derycke, S. & Palomares-Rius, J.E. (2011). Mediterranea 16, 93-99. Genetic structure of Xiphinema pachtaicum and X. index Brown, D.J.F., Halbrendt, J.M., Jones, A.T., Vrain, T.C. & populations based on mitochondrial DNA variation. Phy- Robbins, R.T. (1994). Transmission of three North American topathology 101, 1168-1175. nepoviruses by populations of four distinct species of the Gutiérrez-Gutiérrez, C., Cantalapiedra-Navarrete, C., Decrae- Xiphinema americanum group. Phytopathology 84, 646-649. mer, W., Vovlas, N., Prior, T., Palomares-Rius, J.E. & Castillo, Cherry, T., Szalanski, A.L., Todd, T.C. & Powers, T.O. (1997). P. (2012). Phylogeny, diversity, and species delimitation in The internal transcribed spacer region of Belonolaimus (Ne- some species of the Xiphinema americanum-group complex mata: Belonolaimidae). Journal of Nematology 29, 23-29. (Nematoda: Longidoridae), as inferred from nuclear and mi- Cho, M.R. & Robbins, R.T. (1991). Morphological variation tochondrial DNA sequences and morphology. European Jour- among 23 Xiphinema americanum populations. Journal of nal of Plant Pathology 134, 561-597. Nematology 23, 134-144. He, Y., Subbotin, S.A., Rubtsova, T.V., Lamberti, F., Brown, Conow, C., Fielder, D., Ovadia, Y. & Libeskind-Hadas, R. D.J.F. & Moens, M. (2005a). A molecular phylogenetic ap- (2010). Jane: a new tool for the cophylogeny reconstruction proach to Longidoridae (Nematoda: Dorylaimida). Nemato- problem. Algorithms for Molecular Biology 5, 16. logy 7, 111-124. Coolen, W. (1979). Methods for the extraction of Meloidogyne He, Y., Jones, J., Armstrong, M., Lamberti, F. & Moens, M. spp. and other nematodes from roots and soil. In: Lamberti, (2005b). The mitochondrial genome of Xiphinema america- F. & Taylor, C.E. (Eds). Root-knot nematodes (Meloidogyne num sensu stricto (Nematoda: ): considerable econ- species): systematics, biology and control. London, UK, omization in the length and structural features of encoded Academic Press, pp. 317-329. genes. Journal of Molecular Evolution 61, 819-833. Coomans, A. (1996). Phylogeny of the Longidoridae. Russian Huelsenbeck, J.P. & Ronquist, F. (2001). MRBAYES: Bayesian Journal of Nematology 4, 51-59. inference of phylogenetic trees. Bioinformatics 17, 754-755. Coomans, A. & Claeys, M. (1998). Structure of the female Kim, M., Oh, H.S., Park, S.C. & Chun, J. (2014). Towards a tax- reproductive system of Xiphinema americanum (Nematoda: onomic coherence between average nucleotide identity and Longidoridae). Fundamental and Applied Nematology 21, 16S rRNA gene sequence similarity for species demarcation 569-580. of prokaryotes. International Journal of Systematic and Evo- Coomans, A., Vandekerckhove, T.T. & Claeys, M. (2000). lutionary Microbiology 64, 346-351. Transovarial transmission of symbionts in Xiphinema brevi- Kumari, S. & Subbotin, S.A. (2012). Characterization of Longi- collum (Nematoda: Longidoridae). Nematology 2, 443-449. dorus helveticus (Nematoda: Longidoridae) from the Czech De Grisse, A.T. (1969). Redescription ou modifications de Republic. European Journal of Plant Pathology 133, 923- quelques techniques utilisées dans l’étude des néma- 933. todes phytoparasitaires. Mededelingen Rijksfakulteit Land- Kumari, S., Decraemer, W., De Luca, F. & Tiefenbrunner, bouwwetenschappen Ghent 34, 351-369. W. (2010). Cytochrome c oxidase subunit 1 analysis of Decraemer, W. & Robbins, R.T. (2007). The who, what and Xiphinema diversicaudatum, X. pachtaicum, X. simile and where of Longidoridae and Trichodoridae. Journal of Nema- X. vuittenezi (Nematoda, Dorylaimida). European Journal of tology 39, 295. Plant Pathology 127, 493-499.

Vol. 18(9), 2016 1041 V. Orlando et al.

Lamberti, F. & Bleve-Zacheo, T. (1979). Studies on Xiphinema 1961 and comments on the group. Fundamental and Applied americanum sensu lato with descriptions of fifteen new Nematology 21, 475-490. species (Nematoda, Longidoridae). Nematologia Mediter- Messing, J. (1983). New M13 vectors for cloning. Methods in ranea 7, 51-106. Enzymology 101, 20-78. Lamberti, F. & Ciancio, A. (1993). Diversity of Xiphinema Meza, P., Aballay, E. & Hinrichsen, P. (2011). Molecular americanum-group species and hierarchical cluster-analysis and morphological characterisation of species within the of morphometrics. Journal of Nematology 25, 332-343. Xiphinema americanum-group (Dorylaimida: Longidoridae) Lamberti, F. & Golden, A.M. (1984). Redescription of from the central valley of Chile. Nematology 13, 295-306. Xiphinema americanum Cobb, 1913 with comments on its Navas, A., Fe Andres, M. & Arias, M. (1990). Biogeography of morphometric variations. Journal of Nematology 16, 204- Longidoridae in the Euromediterranean areas. Nematologia 206. Mediterranea 18, 103-112. Lamberti, F. & Siddiqi, M.R. (1977). Xiphinema pachtaicum (= Nunn, G.B. (1992). Nematode molecular evolution: an inves- X. mediterraneum). In: CIH descriptions of plant-parasitic tigation of evolutionary patterns among nematodes based nematodes, Set 7, No. 94. Farnham Royal, UK, Common- upon DNA sequences. Ph.D. Thesis, University of Notting- wealth Agricultural Bureaux. ham, Nottingham. Lamberti, F., Ciancio, A., Agostinelli, A. & Coiro, M.I. (1992). Oliveira, C.M.G., Hübschen, J., Brown, D.J.F., Ferraz, L.C.C.B., Relationships berween Xiphinema brevicolle and X. diffusum Wright, F. & Neilson, R. (2004). Phylogenetic relationships with a redescription of X. brevicolle and description of among Xiphinema and Xiphidorus nematode species from three new species of Xiphinema (Nematoda: Dorylamida). Brazil inferred from 18S rDNA sequences. Journal of Nema- Nematologia Mediterranea 19(1991), 311-326. tology 36, 153-159. Lamberti, F., Molinari, S., Moens, M. & Brown, D.J.F. (2000). Posada, D. (2008). jModelTest: phylogenetic model averaging. Xiphinema americanum The -group. I. Putative species, their Molecular Biology and Evolution 25, 1253-1256. geographical occurrence and distribution, and regional poly- Robbins, R.T. (1993). Distribution of Xiphinema americanum tomous identification keys for the group. Russian Journal of and related species in North America. Journal of Nematology Nematology 8, 65-84. 25, 344-348. Lamberti, F., Molinari, S., Moens, M. & Brown, D.J.F. (2002). Robbins, R.T. & Brown, D.J.F. (1991). Comments on the tax- The Xiphinema americanum-group. II. Morphometric rela- onomy, occurrence and distribution of Longidoridae (Nema- tionships. Russian Journal of Nematology 10, 99-112. toda) in North America. Nematologica 37, 395-419. Lamberti, F., Hockland, S., Agostinelli, A., Moens, M. & Brown, Roca, F., Lamberti, F. & Agostinelli, A. (1989). I Longidori- D.J.F. (2004). The Xiphinema americanum group. III. Keys to dae (Nematoda, Dorylaimida) delle regioni Italiane. IX. La species identification. Nematologia Mediterranea 32, 53-56. Sicilia. Nematologia Mediterranea 17, 151-165. Lazarova, S.S., Malloch, G., Fenton, B., Oliveira, C.M.G., Rob- bins, R.T., Lamberti, F., Brown, D.J.F. & Neilson, R. (2003). Sakai, H., Takeda, A. & Mizukubo, T. (2011). First report of Endosymbiont bacteria of Xiphinema americanum group ne- Xiphinema brevicolle Lordello et Costa, 1961 (Nematoda, matodes. Proceedings of 16th Symposium of Nematological Longidoridae) in Japan. ZooKeys 135, 21-40. Society of South Africa, 1-4 July, 2003, Cape Town, South Seinhorst, J.W. (1959). A rapid method for the transfer of Africa, p. 54. nematodes from fixative to anhydrous glycerin. Nematologica Lazarova, S.S., Brown, D.J.F., Malloch, G., Oliveira, C.M.G., 4, 67-69. Vandekerckhove, T.T.M., Fenton, B., Wright, F., Lamberti, Seinhorst, J.W. (1966). Killing nematodes for taxonomic study F., Barsi, L. & Neilson, R. (2004). Phylogenetic relationships with hot fa 4: 1. Nematologica 12, 178-178. of the endosymbiont bacteris of Xiphinema americanum- Siddiqi, M.R. (1977). Xiphinema mediterraneum Martelli et group nematodes. Proceedings of 27th ESN International Lamberti, a junior synonym of X. pachtaicum (Tulaganov) Symposium, 14-18 June, Rome, Italy, pp. 70-71. Kirjanova. Nematologia Mediterranea 5, 133-135. Lazarova, S.S., Malloch, G., Oliveira, C.M.G., Hubschen, J. & Sites Jr, J.W. & Marshall, J.C. (2004). Operational criteria for Neilson, R. (2006). Ribosomal and mitochondrial DNA anal- delimiting species. Annual Review of Ecology, Evolution and yses of Xiphinema americanum-group populations. Journal Systematics 35, 199-227. of Nematology 38, 404-410. Subbotin, S.A., Stanley, J.D., Ploeg, A.T., Tanha Maafi, Z., Lazarova, S.S., Brown, D.J.F., Oliveira, M.G., Fenton, B., Tzortzakakis, E.A., Chitambar, J.J., Palomares-Rius, J.E., MacKenzie, K., Wright, F., Malloch, G. & Neilson, R. (2016). Castillo, P. & Inserra, R.N. (2015). Characterisation of Diversity of endosymbiont bacteria associated with a non- populations of Longidorus orientalis Loof, 1982 (Nematoda: filarial nematode group. Nematology 18, 615-623. Dorylaimida) from date palm (Phoenix dactylifera L.) in the Luc, M., Coomans, A., Loof, P.A.A. & Baujard, P. (1998). The USA and other countries and incongruence of phylogenies Xiphinema americanum-group (Nematoda: Longidoridae). 2. inferred from ITS1 rRNA and coxI genes. Nematology 17, Observations on Xiphinema brevicollum Lordello & da Costa, 459-477.

1042 Nematology Studies on the Xiphinema americanum-group

Swofford, D.L. (2002). PAUP∗. Phylogenetic analysis using Vrain, T.C. (1993). Restriction fragment length polymorphism parsimony (∗ and other methods), version 4. Sunderland, MA, separates species of the Xiphinema americanum group. Jour- USA, Sinauer Associates. nal of Nematology 25, 361-364. Tanha Maafi, Z., Subbotin, S.A. & Moens, M. (2003). Molecu- Vrain, T.C., Wakarchuk, D.A., Levesque, C.A. & Hamilton, R.I. lar identification of cyst-forming nematodes (Heteroderidae) (1992). Intraspecific rDNA restriction fragment length poly- from Iran and a phylogeny based on the ITS sequences of morphism in the Xiphinema americanum group. Fundamental rDNA. Nematology 5, 99-111. and Applied Nematology 15, 563-573. Taylor, C.E. & Brown, D.J.F. (1997). Nematode vectors of plant Ye, W., Szalanski, A.L. & Robbins, R.T. (2004). Phyloge- viruses. Wallingford, UK, CAB International. netic relationships and genetic variation in Longidorus and Vandekerckhove, T., Willems, A., Gillis, M. & Coomans, Xiphinema species (Nematoda: Longidoridae) using ITS1 se- A. (2000). Occurrence of novel verrucomicrobial species, quences of nuclear ribosomal DNA. Journal of Nematology endosymbiotic and associated with parthenogenesis in 36, 14-19. Xiphinema americanum-group species (Nematoda, Longi- Zasada, I.A., Peetz, A., Howe, D.K., Wilhelm, L.J., Cheam, D., doridae). International Journal of Systematic and Evolution- Denver, D.R. & Smythe, A.B. (2014). Using mitogenomic ary Microbiology 50, 2197-2205. and nuclear ribosomal sequence data to investigate the phy- Vandekerckhove, T.T., Coomans, A., Cornelis, K., Baert, P. & logeny of the Xiphinema americanum species complex. PLoS Gillis, M. (2002). Use of the Verrucomicrobia-specific probe ONE 9, e90035. EUB338-III and fluorescent in situ hybridization for detection of “Candidatus Xiphinematobacter” cells in nematode hosts. Applied and Environmental Microbiology 68, 3121-3125.

Vol. 18(9), 2016 1043