Nematology 18 (2016) 1015-1043 brill.com/nemy Molecular and morphological characterisation of Xiphinema americanum-group species (Nematoda: Dorylaimida) from California, USA, and other regions, and co-evolution of bacteria from the genus Candidatus Xiphinematobacter with nematodes 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 Xiphinema americanum-group is a large species complex containing more than 50 nematode 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 brevicolle, Xiphinema californicum, Xiphinema pachtaicum, Xiphinema rivesi, 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 taxonomy 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