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73Russian Journal of Nematology, 2018, 26 (2), 109 - 113
Molecular characterisation of gall-forming nematodes, Mesoanguina amsinckiae and Anguina danthoniae (Anguinidae: Tylenchida) from
California, USA
1 2 2 3
Ignacio Cid Del Prado Vera , Vladimir N. Chizhov and Sergei A. Subbotin '
'Colegio de Postgraduados, 56230, Montecillo, Mexico 2Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 117071, Moscow, Russia 3Plant Pest Diagnostics Centre, California Department of Food and Agriculture, 3294 Meadowview Road, 95832-1448, Sacramento, CA, USA e-mail: sergei.a. subbotin@gmail.com
Accepted for publication 17 October 2018
Summary. Two gall-forming nematodes, Mesoanguina amsinckiae and Anguina danthoniae, collected in California were molecularly characterised. Phylogenetic relationships of these species with other plant parasitic Anguinidae based on the analysis of the ITS rRNA gene sequences are given as inferred from Bayesian analysis. Mesoanguina amsinckiae parasitising Amsinckia spp. from the family Boraginaceae formed the lineage within the clade with Ditylenchus and Heteroanguina. The position of A. danthoniae is not well resolved with the genus Anguina. The molecular dataset does not contradict the proposal of synonymision of the genus Cynipanguina with Anguina.
Key words: Amsinckia, Anguina, Danthonia californica, ITS-rRNA, Mesoanguina, phylogeny.
The family Anguinidae Nicoll, 1935 includes mycophagous and plant-parasitic nematodes. The latter are obligate specialised parasites of higher plants, mosses and seaweeds on which they often induce swellings and galls (Subbotin & Riley, 2012). Anguinids are found in all continents except for Antarctica. Several species have been found and described from California, USA including Mesoanguina amsinckiae (Steiner & Scott, 1934) Chizhov & Subbotin, 1985 and Anguina danthoniae (Maggenti, Hart & Paxman 1973) Brzeski, 1981.
Mesoanguina amsinckiae was discovered on the host plant Amsinckia intermedia and firstly described by Steiner and Scott (1934) under the name Anguillulina dipsaci var. amsinckiae. Godfrey (1940) referred to the nematode as Ditylenchus dipsaci var. amsinckiae and Filipjev and Schuurmans Stekhoven (1941) named it as Ditylenchus amsinckiae. Thorne (1961) reclassified the nematode as a species of Anguina. Brzeski (1991) and Siddiqi (1996, 2000) also listed this species in this genus. Chizhov and Subbotin (1985) transferred it to the genus Mesoanguina.
Anguina danthoniae was firstly described under the name Cynipanguina danthoniae Maggenti, Hart & Paxman 1973 from California oatgrass, Danthonia californica, from central California coast. The nematode indices spherical galls ranging in size from 0.5 to 3.0 mm and varying in colour from green to straw yellow (Maggenti et al., 1973). Maggenti et al. (1973) proposed a new genus for this species considering that this species differed from Anguina and all other anguinid-like nematodes by having a digit-like process extending from the posterior glandular portion of the pharynx and having spicules with the discrete suture between calamus and lamina. The genus Cynipanguina was synonymised with Anguina by Brzeski (1981). Later this synomisation was supported by Chizhov and Subbotin (1985, 1990), Krall (1991) and Siddiqi (1996, 2000); however, it was rejected by Fortuner and Maggenti (1987).
The goal of this work is to provide molecular characterisations of A. danthoniae and M. amsinckiae collected in California, USA.
© Russian Society of Nematologists, 2018; doi:10.24411/0869-6918-2018-10010
Fig. 1. Leaf galls of Anguina danthoniae (Scale = 0.5
MATERIAL AND METHODS
Nematode populations, DNA extraction, PCR, and sequencing. Dried galls of Danthonia californica with Anguina danthoniae (Fig. 1) collected by the first author from the type locality and dried Amsinckia plant fragments infected by Mesoanguina amsinckiae obtained from the CDFA collection were used in this study. DNA was extracted from several nematodes using proteinase K protocol (Tanha Maafi et al., 2003). The ITS rRNA gene was amplified with the forward TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and the reverse AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') primers as described by Tanha Maafi et al. (2003). PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and directly sequenced. New sequences were deposited in the GenBank under accession numbers: MK032869 and MK032870.
Phylogenetic analysis. Sequences of ITS rRNA genes of M. amsinckiae and A. danthoniae were aligned using Clustal_X 1.83 (Thompson et al., 1997) with published gene sequences of anguinid nematodes (e.g., Powers et al., 2001; Subbotin et al., 2004, 2005; Bertozzi & Davies, 2009; Mobasseri et al., 2017; Barrantes-Infante et al., 2018). Alignment was analysed with Bayesian
inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). Models of DNA evolution for BI were estimated using MrModeltest 2.2 and the best-fit was chosen using the Akaike Information Criterion (Nylander, 2002). BI analysis under the GTR + G model was initiated with a random starting tree and run with the four Metropoliscoupled Markov chain Monte Carlo (MCMC) for 3 x 106 generations. The MCMC were sampled at intervals of 100 generations. After discarding burn-in samples and evaluating convergence the remaining samples were retained for further analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) more than 70% are given for appropriate clades.
RESULTS AND DISCUSSION
The ITS rRNA gene sequence alignment contained 51 sequences including sequences of Orrina phyllobia and Cephalenchus cephalodiscus selected as outgroup species. It was 829 bp in a length. Mesoanguina amsinckiae clustered with a moderate statistical support (PP = 81%) in the clade containing Ditylenchus species and Heteroanguina ferulae. Anguina danthoniae formed a separate lineage and its relationships with other anguinids were unresolved (Fig. 2).
93
Ditylenchus sp.E (Crepis praemorsa, AY574303) - Ditylenchus sp.D (Pilosella caespitosa, AY574304) 60 JL Ditylenchus oncogenus (Sonchus bulbosus, KF612016) 100 I" Ditylenchus taurae (Potamogeton perfoliatus, KX389268) if- Ditylenchus gigas (Vicia fabai, KC310732) I— Ditylenchus weischeri (Cirsium setosum, AF396322) Ditylenchus sp.G (Plantago maritima, AY574287) Ditylenchus dipsaci (Allium sativum, KU179474)
- Heteroanguina ferulae (Ferula Jaeschkeana, AF396324)
- Heteroanguina ferulae (Ferula sp , AF396325)
- Mesoanguina amsinckiae {Amsinckia sp,, California, USA, CD1204, MK03287Ü)
i Mesoanguina picridis (Acroptilon repens, AF396326) Mesoanguina mobilis (Arctotheca calendula, AF396331) Mesoanguina sp. [Anaphalis margaritacea, AF396332) - Heteroanguina caricis {Carex acuta, AF396311}
81
97
90.
90
63 98 98
t
f
H
Afrina wevelli (Eragrostis curvula, KU052862)
- Anguinid nematode (Astrebla pectinata, AF396368)
Afrina sporoboliae (Sporobolus cryptandrus, KM114441) Anguinid nematode (Stipa scabra, AF396316) Heteroanguina graminophila (Calamagrostis neglect a, AF396315) Anguinid nematode (Ageratum conyzoides, KT205559) Litylenchus coprosma (Coprosma repens, GU727548)
Subanguina radicicoia (Poa sp., AF396365) ioo |— Mesoanguina moxae (Artemisia rubripes, AF396314)
Mesoanguina millefolii (Achillea millefolium, AF396312)
■c*
99 97 34 79
32
79
70
£
Ditylenchus askenasyi (Calliergon sp., AF396336) Anguina sp. (Koeleria gracilis, AF396345) Anguina agrostis (Agrostis capiliaris, KM114437) Anguina sp. (Trisetum flavescens, AF396346)
tr
<— Anguina funesta (Lolium rigidum, KM114435) L Anguina sp, (Hotcus lanatus, AF396350) Anguina graminis (Festuca rubra, AF396351) ■vib— Anguina obesa (Alopecurus mysuroides, KX385107) '— Anguina phalaridis (Phleum phieoides, AF396352)
— Anguina sp. (Dactylis glomerata, KM114442) Anguina agropyronifloris (Agropyron smithii, AF363093)
Anguina tritici (Tríticum sp,, KT900693) ■ Anguina sp. (Poa malacantha, AF396360) - Anguina sp. (Hierochloe alpina, AF396359) Anguina sp. (Arctagrostis latifolia, AF396356) L Anguina sp (Calamagrostis purpurea, AF396358) L Anguina sp. {Festuca nigrescens, AF396362)
— Anguina paludicota (Polypogon monspeliensis, AF396363) Anguina pacificae (Poa sp., KM114440) Anguina agropyri (Elymus repens, AF396355)
100 |- Anguina australis (Ehrharta longifiora, AF396334)
H.:
L Anguina woodii (Ehrharta villosa, AY307122) -Anguina danthoniae (Danthonia californica, California, USA, CD12Ü1, MK032869)
— Anguina microiaenae (Microlaena stipoides, AF396333)
--Cephalenchus cephalodiscus (KX462090)
- Orrina phyllobia (Solanum elaegnifolium, KT192616)
0.1
Fig. 2. Phylogenetic relationships within some species of Anguinidae as inferred from Bayesian analysis using the ITS rRNA gene sequence data set under the GTR + G model. Posterior probabilities of over 70% are given for appropriate clades. Newly obtained sequences are indicated in bold.
Topology of the obtained tree is congruent with those published by Subbotin et al. (2004) and Barrantes-Infante et al. (2018). Subbotin et al. (2004) noticed a paraphyly of the genus Mesoanguina parasitising Asteraceae and found that the studied species of this genus clustered into two distinct groups in an agreement with their peculiarities of morphology and host plant range. In
our tree the genus Mesoanguina parasitising Asteraceae is also divided in these two groups; moreover, M amsinckiae parasitising Amsinckia spp. from the family Boraginaceae formed the lineage within the clade with Ditylenchus and Heteroanguina. Thus, the revision of the non-monophyletic genus Mesoanguina is still needed to transfer these groups in different taxon units.
The genus Anguina is monophylic according to Subbotin et al. (2004) and Barrantes-Infante et al. (2018). Two species, A. australis and A. microlaenae inducing leaf and stem galls in plants of the subfamily Bambusoideae, occupied a basal position within Anguina species parasitising Poaceae in the tree published by Subbotin et al. (2004) and Barrantes-Infante et al. (2018). In the present tree the positions of A. australis plus A. woodi and A. microlaenae as well as A. danthoniae are not resolved with the genus Anguina. However, the monophyly of the genus Anguina including A. danthoniae is not rejected based on this ITS rRNA topology. Thus, the molecular dataset does not contradict the proposal to synonymise the genus Cynipanguina with Anguina made by Brzeski (1981).
Comprehensive morphological and molecular analyses are still needed to understand relationships within anguinid nematodes and replace the present artificial classification by a natural one reflecting evolutionary trends in this group (Subbotin & Riley, 2012).
ACKNOWLEDGEMENTS
This work was supported by a FarmBill19 grant, CDFA Agmt no. 18-0346-000-FR.
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Cid Del Prado Vera, I., V.N. Chizhov and S.A. Subbotin. Молекулярная характеристика галлообразующих нематод, Mesoanguina amsinckiae и Anguina danthoniae (Anguinidae: Tylenchida) из Калифорнии, США.
Резюме. Приводятся данные по нуклеотидным последовательностям двух видов галлообразующих нематод Mesoanguina amsinckiae и Anguina danthoniae, обнаруженных в Калифорнии, США. По результатам Байесова анализа последовательностей ITS рДНК определены филогенетические взаимоотношения этих видов с другими паразитирующими на растениях видов семейства Anguinidae. Вид Mesoanguina amsinckiae, паразитирующий на Amsinckia spp. из семейства Boraginaceae, показал родство с родами Ditylenchus и Heteroanguina. Взаимоотношения вида A. danthoniae с другими видами рода Anguina остаются неразрешенным. Молекулярные данные не противоречат предложенной синонимизации рода Cynipanguina с Anguina.
