1 ZOOL J LINN SOC……… 1 1 2Integrative taxonomy unravels cryptic diversity in the Paratrichodorus hispanus-group 3complex to resolve two new species of the genus and the molecular phylogeny of the 4family (Nematoda: Trichodoridae) 5 6 7Wilfrida Decraemer1, Carolina Cantalapiedra-Navarrete2, Antonio Archidona-Yuste2, Ingrid 8Varela-Benavides3, Carlos Gutiérrez-Gutiérrez4,5, Pablo Castillo2, and Juan E. Palomares-Rius2,* 9 101Ghent University, Department of Biology, Ledeganckstraat 35, B-9000 Ghent, Belgium 112Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. 12Menendez Pidal s/n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, 13ceiA3, Spain 143Laboratorio de Nematología, Instituto Tecnológico de Costa Rica sede San Carlos, Apartado 15postal: 223-21001, Alajuela, San Carlos, Costa Rica 164NemaLab/ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas & Dept. de 16 17Biologia, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal 185 Dept. Protección Vegetal, Estación Experimental de Santa Catalina, Instituto Nacional 19Autonómico de Investigaciones Agropecuarias (INIAP), Panamericana Sur s/n, Ap. 1701-340, 20Machachi, Ecuador/Project Prometeo - SENESCYT, Quito 170184, Ecuador 21 22*Corresponding author. E-mail: [email protected] 23 24Running Head: Paratrichodorus hispanus-group complex 25 26Received ….; revised ….; accepted for publication ……. 27 28 1 ZOOL J LINN SOC……… 2 1The genus Paratrichodorus currently comprises 26 species of polyphagous 2ectoparasitic plant nematodes that are distributed worldwide and include a few 3tobravirus vector species. Paratrichodorus is one of the most difficult genera in terms of 4species identification because it is phenotypically conserved with morphometric 5characters that often overlap between species and specimens are highly sensitive to 6fixatives. From 2003 to 2016, nematode surveys were conducted in southern Spain, 7and the current contribution addresses 22 nematode populations of the 8Paratrichodorus hispanus-group. Among them, two new species Paratrichodorus 9almadenensis sp. nov. and Paratrichodorus ramblensis sp. nov., are described 10here morphologically and molecularly and compared with topotype specimens of P. 11hispanus. Furthermore, this analysis was complemented with the first molecular data of 12this species complex as well as for the genus Monotrichodorus. The phylogeny of 13family Trichodoridae showed the relationship of Monotrichodorus with Trichodorus, both 14of which are phylogenetically associated with the P. hispanus species group, including 15the two newly described species. Several phylogenetic hypotheses about the 16monophyly of the genera with molecular data composing the family Trichodoridae were 17tested using Shimodaira-Hasegawa and approximately unbiased tests. This work also 18includes the first observations of copulatory plugs in Paratrichodorus, which are of a 19unique type, which clearly differs from the different types of mating plugs described in 20Trichodorus species. 21 22 23 24 25 26 27 28ADDITIONAL KEY WORDS: Bayesian inference, cytochrome oxidase c subunit 1 29(coxI), cryptic species, stubby root nematodes, approximately unbiased and 30Shimodaira-Hasegawa tests, expansion domains of the large ribosomal subunit (28S), 31internal transcriber spacer, rRNA 32 1 ZOOL J LINN SOC……… 3 1INTRODUCTION 2Stubby root nematodes of the family Trichodoridae include 111 species grouped within 3six genera including three didelphic genera and three monodelphic genera (Decraemer, 41995; Decraemer & Robbins, 2007). Paratrichodorus is the second most species-rich 5didelphic genus (26 species) after Trichodorus (65 species), but seven of the species in 6the former have been transferred to the genus Nanidorus upon the recognition of the 7latter as a valid genus (Duarte et al., 2010). The other monodelphic-prodelphic genera 8are Monotrichodorus (5 spp.), Allotrichodorus (6 spp.) and Ecuadorus (2 spp.) that are 9restricted to an area in Central America and the northern part of South America 10(Decraemer & Robbins, 2007). 11 Within the family Trichodoridae, identification to the species level is hampered by 12the largely conserved morphology, overlapping morphometrics and co-occurrence of 13more than one trichodorid species in the same soil sample. Another issue is the 14difficulty in properly fixing trichodorid nematodes, particularly Paratrichodorus species, 15as well as the degradation of type specimens in nematode collections that result in 16outer and/or inner disturbance of structures and systems, particularly to the pharynx 17including the stylet (onchium and onchiophore) and the spicules. One more restriction 18includes the need for specimens to be in a lateral position to correctly observe the main 19diagnostic features, such as the shape of the vagina and sclerotized pieces in the 20female and spicule shape in the male. 21 The current contribution focuses on the genus Paratrichodorus in the framework 22of continuous surveys for Trichodoridae in cultivated and natural environments in 23southern Spain since 2003. A previous paper on the genus Trichodorus, including 24seven new species, was published by Decraemer et al., (2013).. The main diagnostic 25morphological characters for Paratrichodorus at the species level are: (i) in males, the 26shape, ornamentation and length of the spicules, the number and position of precloacal 27supplements within the region of retracted spicules and the number and position of 28ventromedian cervical papillae and to a lesser extent tail shape/length, the position of 29the postcloacal papillae and the size of the bursa; and (ii) in females, the size, shape, 30position and orientation of the vaginal sclerotized pieces (= pars refringens vaginae) in 31optical section of the sclerotized ring and to a lesser extent the size and shape of the 32vagina and the presence of sublateral body pores. Since the information provided by 33morphological features may be rather limited, morphometric data such as body length 34and the lengths of the onchiostyle and spicule are more important as diagnostic 35features than in Trichodorus (Decraemer, 1995). However, morphometric data within 1 ZOOL J LINN SOC……… 4 1Paratrichodorus should be considered with caution since fixation result in contraction of 2the outer body cuticle and/or the inner organs (Sturhan, 1985). 3 López-Pérez et al. (2001) revised the Trichodoridae in Spain and found eighteen 4species in the Iberian Peninsula and Islands, including six Paratrichodorus species: P. 5allius (Jensen, 1963) Siddiqi, 1974; P. anemones (Loof, 1965) Siddiqi, 1974; P. 6hispanus Roca & Arias, 1986; P. pachydermus (Seinhorst, 1954) Siddiqi, 1974; P. 7porosus (Allen, 1957) Siddiqi, 1974; and P. teres (Hooper, 1962) Siddiqi, 1974 as well 8as three Nanidorus species: Nanidorus acutus (Bird, 1967) Siddiqi, 1974, N. minor 9(Colbran, 1956) Siddiqi, 1974; and N. nanus (Allen, 1957) Siddiqi, 1974. 10Paratrichodorus teres and N. minor were only recorded from the Canary Islands, and a 11previous report of Paratrichodorus cf. acutus could not be confirmed. Paratrichodorus 12hispanus appeared widespread in Spain and northern Portugal while the other species 13were strongly localised (viz. P. allius and P. anemones), and P. porosus was only found 14in the Azores and Madeira Archipelagos. Duarte et al. (2010) reported sixteen 15trichodorid species from Portugal, including Paratrichodorus divergens Almeida, 16Santos, Abrantes & Decraemer, 2005 as the most recently described species of this 17genus in Portugal. The authors also recorded the presence of P. porosus in continental 18Europe for the first time. Paratrichodorus teres has been recorded in mainland 19Portugal. Paratrichodorus anemones and P. pachydermus are characteristic of 20temperate environments; they are widespread in the UK and western and northern 21Europe. At present, seven out of 26 Paratrichodorus species, or 27%, have been 22recorded in the Iberian Peninsula. The major problem posed by nematodes of 23Trichodoridae to crops is that they are vectors of tobraviruses. Currently, nine 24Paratrichodorus species (26%) are known to be natural tobravirus vectors (Decraemer 25& Robbins 2007). Except for P. porosus, all Paratrichodorus species known at present 26from the Iberian Peninsula are natural vectors of these plant viruses. 27 Molecular characterization based on 18S rRNA gene by Kumari & Subbotin 28(2012) showed two main clades of Paratrichodorus species: (i) clade 1 with two 29subclades, one with species with undulating spicular calomus (all present in the Iberian 30Peninsula, P. anemones, P. hispanus complex, Paratrichodorus sp. B, Paratrichodorus 31sp. C, and P. divergens) and one with two species frequent in temperate latitudes with 32non-undulating spicules (P. pachydermus and P. macrostylus), and (ii) clade 2 with the 33species P. allius, P. porosus and P. teres for which males can be rare; the first two 34species are more common in warmer climates. This observation confirms the 35subdivision found by Duarte et al. (2010) based on the 18S rRNA gene. 1 ZOOL J LINN SOC……… 5 1 Hitherto, no studies with molecular data illustrate the phylogenetic relationship 2between the monodelphic and didelphic genera in the family Trichodoridae that share 3important morphological features. For example, the variable swelling of the body cuticle 4and an approximately straight body appearance as well as the presence of a bursa and 5copulatory muscles restricted to the spicule region