Nematoda: Tylenchomorpha)

Identification, systematics and phylogeny of the genera in the family Aphelenchoididae (Nematoda: Tylenchomorpha)

Yiwu Fang

Promoters: Prof. Hongmei LI and Prof. Wim BERT

Academic year 2017-2018

Thesis submitted in fulfillment of the requirements for the degree of Doctor in Science, Biology

Examination Committee

Prof. Hongmei Li Promotor, Nanjing Agricultural University, Nanjing, China

Prof. Wim Bert Promotor, Ghent University, Belgium

Prof. Wilfrida Decraemer Secretary, Ghent University, Belgium

Prof. Jingwu Zheng Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

Dr. Jianfeng Gu Technical Centre, Ningbo Entry-exit Inspection and Quarantine Bureau, Ningbo, China

Table of contents

Acknowledgements ...... i Summary ...... iii Samenvatting ...... vii 摘要 ...... viii Chapter I: General Introduction ...... 1 Classification ...... 2 Ecology and plant-parasitism of Aphelenchoididae ...... 5 General morphology ...... 8 BODY HABITUS ...... 8 HEAD REGION ...... 10 CUTICLE AND LATERAL REGION ...... 10 STYLET ...... 11 PHARYNX...... 12 MALE COPULATORY SYSTEM ...... 15 TAIL ...... 17 Molecular identification and phylogeny of Aphelenchoididae ...... 18 OBEJECTIVES AND THESIS OUTLINE ...... 22 Chapter II: Description of Aphelenchoides stellatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from Japan ...... 25 Abstract ...... 26 Introduction ...... 27 Materials and methods ...... 27 CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS ...... 27 MOLECULAR STUDIES ...... 27 Results ...... 28 MEASUREMENTS ...... 28 DESCRIPTION ...... 28 TYPE HABITAT AND LOCALITY ...... 33 TYPE MATERIAL ...... 33 MOLECULAR CHARACTERISATION AND PHYLOGENY ...... 35 Discussion ...... 38 Chapter III: Description of Aphelenchoides rotundicaudatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from South Korea ...... 39 Abstract ...... 40 Introduction ...... 41 Materials and methods ...... 41 CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS ...... 41 MOLECULAR STUDIES ...... 41 Results ...... 42 MEASUREMENTS ...... 42 DESCRIPTION ...... 42 TYPE HABITAT AND LOCALITY ...... 47 TYPE MATERIAL ...... 47 DIAGNOSIS AND RELATIONSHIPS ...... 47 MOLECULAR CHARACTERISATION AND PHYLOGENY ...... 48 Discussion ...... 52 Chapter IV: Description of Sheraphelenchus parabrevigulonis n. sp. (Nematoda: Aphelenchoididae) in pine wood packaging from Italy and redescription of S. sucus in onion bulbs from South Korea, isolated at Ningbo, China ...... 55 Abstract ...... 56 Introduction ...... 57 Materials and methods ...... 57 CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS ...... 57 MOLECULAR STUDIES ...... 57 Results ...... 58 Sheraphelenchus parabrevigulonis n. sp...... 58 MEASUREMENTS ...... 58 DESCRIPTION ...... 58 TYPE HABITAT AND LOCALITY ...... 64 TYPE MATERIAL ...... 64 DIAGNOSIS AND RELATIONSHIPS ...... 64 S. sucus Kanzaki & Tanaka, 2013 ...... 65 MEASUREMENTS ...... 65 DESCRIPTION ...... 65 VOUCHER MATERIAL ...... 70 DIAGNOSIS AND RELATIONSHIPS ...... 71 MOLECULAR CHARACTERISATION AND PHYLOGENY ...... 71 Discussion ...... 75 Chapter V: Description of Pseudaphelenchus zhoushanensis n. sp. (Tylenchina: Aphelenchoididae) found in the wood of Pinus thunbergii at Zhoushan Islands, Zhejiang Province, China ...... 79 Abstract ...... 80 Introduction ...... 81 Materials and methods ...... 81 CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS ...... 81 MOLECULAR ANALYSES ...... 82 Results ...... 83 MEASUREMENTS ...... 83 DESCRIPTION ...... 83 TYPE HABITAT AND LOCALITY ...... 89 TYPE MATERIAL ...... 90 DIAGNOSIS AND RELATIONSHIPS ...... 90 MOLECULAR PROFILES AND PHYLOGENETIC STATUS ...... 91 Discussion ...... 92 Chapter VI: Description of five unknown Aphelenchoides species ...... 97 Abstract ...... 98

Introduction ...... 99 Materials and methods ...... 99 CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS ...... 99 MOLECULAR ANALYSES ...... 100 Results ...... 101 Aphelenchoides sp. 12Asp ...... 102 MEASUREMENTS ...... 102 DESCRIPTION ...... 102 DIAGNOSIS AND RELATIONSHIPS ...... 107 Aphelenchoides sp. 14Asp ...... 109 MEASUREMENTS ...... 109 DESCRIPTION ...... 109 DIAGNOSIS AND RELATIONSHIPS ...... 113 Aphelenchoides sp. 18Asp ...... 117 MEASUREMENTS ...... 117 DESCRIPTION ...... 117 Aphelenchoides sp. AspX ...... 125 MEASUREMENTS ...... 125 DESCRIPTION ...... 125 DIAGNOSIS AND RELATIONSHIPS ...... 129 Aphelenchoides sp. Asp25 ...... 133 MEASUREMENTS ...... 133 DESCRIPTION ...... 133 DIAGNOSIS AND RELATIONSHIPS ...... 137 MOLECULAR PROFILES AND PHYLOGENETIC STATUS ...... 140 Discussion ...... 147 Chapter VII: Mapping head region characteristics on the molecular phylogeny of Aphelenchoididae ...... 151 Abstract ...... 152 Materials and methods ...... 154 NEMATODE CULTURING AND MORPHOLOGICAL OBSERVATIONS ...... 154 MOLECULAR ANALYSES ...... 156 Results ...... 157 CHARACTERIZATION OF THE HEAD PATTERNS OF THE GENERA IN THE FAMILY APHELENCHOIDIDAE ...... 157 Genus Aphelenchoides ...... 160 Genus Aprutides ...... 161 Genus Bursaphelenchus ...... 161 Genus Cryptaphelenchus ...... 162 Genus Devibursaphelenchus ...... 163 Genus Ficophagus ...... 163 Genus Laimaphelenchus...... 164 Genus Pseudaphelenchus ...... 164 Genus Robustodorus ...... 164 Genus Ruehmaphelenchus ...... 165 Genus Schistonchus ...... 165 Genus Seinura ...... 165 MOLECULAR PROFILES AND PHYLOGENETIC STATUS ...... 166 PHYLOGENETIC STATUS AND HEAD REGION CHARACTERS ...... 175 Cephalic sectors ...... 183 Oral aperture ...... 183 Inner labial sensilla ...... 184 Outer labial sensilla ...... 184 Cephalic sensilla ...... 184 Amphids apertures ...... 184 Chapter VIII: General discussion and conclusion ...... 195 Taxonomy and phylogeny in Aphelenchoididae ...... 196 Ecology diversity in Aphelenchoididae ...... 197 Mapping head region characteristics in Aphelenchoididae ...... 199 References ...... 201 List of Publications...... 227

Acknowledgements

My thesis work has received the financial support by the Natural Science Foundation of China (Grant No. 31471751), and the special research fund UGent 01N02312. I want to thank the Chinese Scholarship Council (CSC) (No. 201506850062) for providing me a research fellowship study at Ghent University, Belgium.

Foremost, I would like to extend my sincere gratitude to my supervisors, Profs Dr. Hongmei Li and Dr. Wim Bert, for their both professional and personal guidance and continuous support to my PhD study and research. This thesis would not have been possible without their efforts. Their ideas and enthusiasm for nematology will be a great treasure in my research life forever. I cannot find words to express my gratitude to Prof Hongmei Li who gave me the life guidance from the beginning to the end of my PhD study, she is my teacher forever.

My sincere thanks to the four years time in the Plant Nematology Lab of Nanjing Agricultural University. I am grateful to Dr. Xuan Wang, who gave me insightful comments and encouraged my study and research, and to other the-current and former-members, who contributed immensely to my personal and professional time: Xiuhu Le, Yuankai Chi, Bingliang Liu, Chenggang Sun, Pei Wang, Yuliang Ju, Yu Lin, Zhiqiang Song, Guilin Xiang, Cunjun Liu, Rui Hao, Feifei Gao, Tinglong Guan, Xudong Liang, Zhuran Qi, Ning Wang, Tingting Wang, Xiaobin Hu, Long Zhang, Hongyan Wei, Hong Li, Hairun Zhu, Maria Munawar, Jinhua Shen, Wenwen Niu, Wenrong Sun, Rongrong Liu, Yang Fa, Yishi Su, Wenwen Wan, Hailu Liu, Juntao Dai, Bowen Xue, Jukui Ma, Zhen Wang, Xin Qin, Linshuo Liu, Xiuheng Ding, Yanxia Li, Xiang He, Renpeng Zhou, Jiarong Yu, Jingjing Cai, Yadong Wang, Qianqian Wang. Thank you all for your company and help in my study and research journey.

I would like to express my gratitude to the one year study in the Nematology Research Unit at Ghent University. I am grateful to the members in the lab who contributed immensely to my personal and professional time: Marjolein Couvreur, Barbara Verstraeten, my colleagues Qing Xue, Alcides Sánchez-Monge, Toon Janssen, Dieter Slos, Yao Kolombia, Daniel Apolonio, Inge Dehennin, Hanne Steel and Giselle Herren who shared their knowledge and advices in i several topics, related or not to nematodes. I would like to thank Prof. Dr. Wilfrida Decraemer and Nic Smol (Ghent University), who brought me to a new nematology world which I didn’t know before. I want to express my very special gratitude to Dr. Qing Xue who gave me huge assistance to live in Ghent. It was a really nice but short journey in Ghent. I love it and thank you all.

I extend my thanks to Dr. Jianfeng Gu, who gave me kind assistance of samples and material for study, his knowledge and enthusiasm for nematology also support my PhD study and research.

I would like to give my special thanks to my classmates, Yang Wang, Ning Ding, Jia Li, Wu Xiong and Landa Qi for your company. May our friendship last forever.

I would like to extend my thanks to all my teachers, classmates and friends and relatives for all their guidance, care, support and help.

Last but not least, I want to thank my parents who gave me selfless love and infinite encouragement to support everything in my life. May Buddha bless you.

Summary

The nematode family Aphelenchoididae Skarbilovich, 1947 includes numerous species with morphological and ecological diversity. Among them, the pine wood nematode Bursaphelenchus xylophilus and the rice white tip nematode Aphelenchoides besseyi can cause serious damage to forestry and agriculture respectively; therefore, they attract great attention from scientists worldwide. However, most of the genera in Aphelenchoididae are not yet well studied, not well delinated and comprise problems from several points of view. In this study, ten species from three genera in Aphelenchoididae isolated from different habitats were characterized and described by morphological and molecular traits. The results enriched the taxonomic information for this family. Additionally, by plotting morphological characters of the head region of Aphelenchoididae species on a Bayesian concatenated tree based on two ribosomal DNA (rDNA) loci illuminated the phylogenetic evolution relationship of members in the family Aphelenchoididae. The results are as following:

1. Description of Aphelenchoides stellatus n. sp.

Aphelenchoides stellatus n. sp. was isolated from packaging wood from Japan imported to Ningbo Port, China. The new species is characterised by a body length of 485-533 μm (males) and 547-699 μm (females). The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 9-11 μm long and has small basal swellings. The excretory pore is located posterior to the nerve ring. Spicules smoothly curved, rose-thorn shaped. Apex and rostrum round, only slightly offset, dorsal limb 19-21 μm long. Male tail bearing six (2 + 2 + 2) caudal papillae. The female spermatheca is axial and oblong with round sperm present in multiple rows. Both male and female tail pegs have 3-4 processes, appearing star-like under the scanning electron microscopy (SEM). The new species belongs to the Group 3 category sensu Shahina (1996) of Aphelenchoides species. Phylogenetic analyses based on full length ITS and 28S D2-D3 region of rDNA confirmed its status as a new species.

2. Description of Aphelenchoides rotundicaudatus n. sp.

Aphelenchoides rotundicaudatus n. sp. was isolated from packaging wood from South Korea. The new species is characterised by a body length of 364-509 μm (males) and 371-493 μm iii

(females). The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 8-9 μm long and has small basal swellings. The excretory pore is located ca one body diam. anterior to median bulb. Spicules are small (10-13 μm), apex and rostrum rounded and only slight offset. The male tail has six (2 + 2 + 2) caudal papillae. Both male and female tails are cylindrical with a broadly rounded terminus, often with a small region of thickened cuticle or a blunt peg ca 1 μm long, particularly in the female. The new species belongs to the Group 1 category of Aphelenchoides species. Phylogenetic analyses based on partial 18S, 28S D2-D3 and full length ITS of rDNA confirmed its status as a new species.

3. Description of Sheraphelenchus parabrevigulonis n. sp. and redescription of S. sucus

Sheraphelenchus parabrevigulonis n. sp. was isolated from pine wood packaging from Italy. The new species is characterised by a stylet length of 11 μm, three lines in the lateral field, excretory pore at level with, or slightly posterior to, nerve ring, V = 93.1, and spicules with long condyles and membrane-like rostrum in a shallow triangular shape. Another Sheraphelenchus population was isolated from onion bulbs from South Korea. The species was identified as S. sucus, which is characterised by a stylet length of 13 μm, excretory pore at level of, or posterior to, median bulb, and V = 80.9. The phylogeny trees based on rDNA 18S, 28S D2-D3 and ITS full length sequences confirmed S. parabrevigulonis n. sp. as a new species and the correct identification of S. sucus.

4. Description of Pseudaphelenchus zhoushanensis n. sp.

Pseudaphelenchus zhoushanensis n. sp. was isolated from a dead Pinus thunbergii at Changgang Mountain, Zhoushan Islands, Zhejiang Province, China. The new species is characterised by the small to medium body length body, cuticle slightly annulated, presence of three lateral lines, stylet 9.0-10.7 μm with small but conspicuous basal knobs, excretory pore located from same level as the metacorpus to slightly anterior to metacorpus, true bursa surrounding entire tail but inconspicuous, male tail conical with a single mucron, spicule with distinct condylus and rostrum strongly arcuate to a pointed end, female tail conical with annulation, strongly ventrally bent in distal part of tail, with terminus bluntly pointed or finely

iv mucronate. Phylogenetic analyses using sequences of the 18S and 28S D2-D3 regions of rDNA confirmed the status of P. zhoushanensis n. sp. as a new species. Combining the molecular phylogenetic analyses, morphology and biology of P. zhoushanensis n. sp. and Tylaphelenchus jiaae indicates that T. jiaae is a member of Pseudaphelenchus to which it is herein transferred as P. jiaae n. comb. (= T. jiaae).

5. Description of five unknown Aphelenchoides species

Six Aphelenchoides populations were isolated from the different habitats in several countries and identified as five unknown species by morpholgical and molecular characters, including Aphelenchoides sp. 12Asp, Aphelenchoides sp. 14Asp, Aphelenchoides sp. 18Asp, Aphelenchoides sp. AspX and Aphelenchoides sp. Asp25. All the unknown species belong to the Group 2 according to the category of Aphelenchoides species of which the female tail has a single mucro. The male of Aphelenchoides sp. 18Asp is characterised by the spicules with bifurcated distal tip, which is a rare character and unknown in Aphelenchoides species. Group 2 comprise a large number of species which make it difficult to distinguish the five unknown speices from the close related species is rather difficult. The phylogenetic analysis of Aphelenchoides species based on rDNA 18S and 28S D2-D3 sequences confirmed the status of these five species as unkown and undescribed new species relative to the known molecular data.

6. Morphology of the head region and phylogenetic relationships of Aphelenchoididae

The characteristics of the head region from six Aphelenchoides populations were observed using SEM. In total, 40 types of head regions were presented based on 78 species from 11 genera of the family Aphelenchoididae. This was based on a comparison of the head region characteristics, including oral aperture, inner labial sensilla, outer labial sensilla, cephalic sensilla, amphids apertures and annulations of head region. The concatenated phylogenetic analyses by combining 404 MOTUs of rDNA 18S and 28S D2-D3 revealed five major clades within Aphelenchoididae. Using the the head region charteristics it is possible to distinguish several closed species, including Aphelenchoides besseyi and A. fujianensis. However, head

v region types in the family Aphelenchoididae did not correspond to phylogenetic relationships, i.e. the major clades. The molecular and SEM characterisations of the members in Aphelenchoididae need to be further explored, and the phylogenetic realtionship in different types of the head regions for the members still need to be clarified.

KEY WORDS: Aphelenchoididae; Aphelenchoides; Sheraphelenchus; Pseudaphelenchus; morphology; morphometrics; new species; head region; scanning electron microscopy; molecular phylogeny; concatenated analysis

Samenvatting

De familie Aphelenchoididae Skarbilovich, 1947, behorend tot het fylum Nematoda, bevat veel soorten en is morfologisch zeer divers. De dennenboomnematode, Bursaphelenchus xylophilus, en de “witte tip” rijstnematode, Aphelenchoides besseyi, kunnen grote schade veroorzaken in de bosbouw en in de landbouw en zijn daarom grondig bestudeerd. De meeste andere soorten daarentegen zijn echter nog onvoldoende gekarakteriseerd en roepen nog veel onduidelijkheden op. In deze studie werden diverse soorten van drie genera uit divers habitatten geïsoleerd en vervolgens morfologische en moleculair en gekarakteriseerd. Negen soorten bleken nieuw voor de wetenschap: Aphelenchoides stellatus n. sp., Aphelenchoides rotundicaudatus n. sp., Sheraphelenchus parabrevigulonis n. sp., en Pseudaphelenchus zhoushanensis n. sp. werden formeel beschreven en vijf overige soorten werden gekarakteriseerd maar nog niet formeel beschreven. De soort S. sucus werd herbeschreven. Deze resultaten verbeteren de taxonomische kennis van de Aphelenchoididae. Op basis van nieuwe sequenties en GenBank rDNA 18S and 28S D2-D3 sequenties (404 molecular taxonomical units in totaal) werd een concatenatie alignement opgesteld. De resulterende fylogenetische boom bevatte 5 duidelijke belangrijke clades. Door morfologische eigenschappen, verkregen via scanning elektronenmicroscopie van de kop regio, te plotten op een fylogenetische boom werden de evolutive relaties binnen de Aphelenchoididae verder geanalyseerd. Hoewel details van de kopregio belangrijk bleken te zijn om bepaalde soorten af te bakenen zoals Aphelenchoides besseyi en A. fujianensis, is er geen duidelijke overeenkomst tussen de evolutie van de kopstructuren en de moleculaire fylogenie.

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摘要

滑刃科线虫 Aphelenchoididae Skarbilovich, 1947 是一类具有高度生态多样性和形态多样性的类群，其中的松材线虫 Bursaphelenchus xylophilus 和贝西滑刃线虫 Aphelenchoides besseyi 可引起重要的植物病害，受到了广泛的关注，但对滑刃线虫科其他成员的研究较少。本文对不同生境中分离出的滑刃线虫科 3 属 10 个种群进行了鉴定和描述，并结合头区形态和贝叶斯法构建的合一系统进化树对滑刃线虫科进行了系统的研究，旨在丰富滑刃线虫科的分类学信息，揭示该科成员的系统进化关系。主要研究结果如下：

1. 星状滑刃线虫新种的鉴定

从日本进境松木包装中分离到滑刃线虫属 Aphelenchoides 一群体，根据雌虫和雄虫的尾尖突呈星状特征，将该群体命名为星状滑刃线虫新种 A. stellatus n. sp.，主要形态鉴别特征为：雌虫、雄虫体长分别为 547-699 μm 和 485-533 μm；体壁环纹细，侧区具 4 条侧线；口针长 9-11 μm 且有明显的基部膨大；排泄孔位于神经环后侧；交合刺呈光滑弯曲的玫瑰刺状，基顶和喙突轻度突出且呈圆形，外缘长 19-21 μm；雄虫尾部有 6 个（2+2+2）尾乳突；雌虫受精囊呈轴向长方形，且包含多排的圆形精子；雌虫、雄虫的尾尖突均有 3~4 个钉状突起，在扫描电子显微镜（SEM）下呈现星状。该新种归属于 Shahina（1996）的滑刃线虫属第 3 组。基于 rDNA 的 ITS 全长和 28S D2-D3 区的系统进化分析亦证实该新种的地位。

2. 圆尾滑刃线虫新种的鉴定

从韩国进境松木包装中分离到滑刃线虫属 Aphelenchoides 一群体，根据雌虫和雄虫的尾端呈圆形特征，将其命名为圆尾滑刃线虫新种 A. rotundicaudatus n. sp.。主要形态特征为：雌虫和雄虫体长分别为 371-493 μm 和 364-509 μm；体壁环纹细，侧区有 4 条侧线；口针长 8-9 μm，有明显的基部膨大；排泄孔位于中食道球前一个体宽长处；交合刺小，弦长 10-13 μm，基顶和喙突轻度突出，呈圆形；雄虫尾部有 6 个（2+2+2）尾乳突；雌虫和雄虫尾形均呈圆柱形，特别是雌虫的尾端宽圆，但尾末端的角质层有加厚，或有一个长约 1 μm 的钝突。该新种归属于滑刃线虫属第 1 组。基于 rDNA 18S、ITS 和 28S D2-D3 序列的系统进化分析证实该种为一新种。

3. 拟短食道希尔滑刃线虫新种的鉴定和树液希尔滑刃线虫的重新描述

从意大利进境松木包装材料中分离到希尔滑刃线虫属 Sheraphelenchus 一群体，根据其形态特征与短食道希尔滑刃线虫 S. brevigulonis 相似，将该群体命名为拟短食道希

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尔滑刃线虫新种 S. parabrevigulonis n. sp.，主要鉴别特征为：口针长 11 μm；侧区有 3 条侧线；排泄孔位于神经环后侧；阴门异常靠后，V 值 = 93.1；交合刺有长基顶和三角形的翼膜状喙突。从韩国进境洋葱球茎中分离到 Sheraphelenchus 另一群体，将其鉴定为树液希尔滑刃线虫 S. sucus，主要鉴别特征为：口针长 13 μm；排泄孔位于中食道球等水平或略靠后；阴门靠后，V 值 = 80.9。基于 rDNA 18S、ITS 和 28S D2-D3 区序列的系统进化分析证实了拟短食道希尔滑刃线虫的新种地位和树液希尔滑刃线虫的鉴定正确。

4. 舟山假滑刃线虫新种的鉴定

从浙江舟山长岗山黑松死树上分离到假滑刃属线虫 Pseudaphelenchus 一群体，根据发现地将其命名为舟山假滑刃线虫新种 P. zhoushanensis n. sp.，主要鉴别特征为：体形中等，体壁环纹细；侧区有 3 条侧线；口针长 9.0-10.7 μm，口针基部球小但明显；排泄孔位于中食道球瓣门等水平处或至略靠中食道球前缘处；交合伞包裹雄虫尾，但不明显；雄虫尾部圆锥形，有单个尾尖突；交合刺基顶和喙突明显，喙突强烈弯曲，形成一个尖端；雌虫尾圆锥形，有环纹，在近末端处向腹面强烈弯曲，尾端钝尖，或有一个精细的尾尖突。基于 rDNA 18S 和 28S D2-D3 序列构建的系统进化分析证实舟山假滑刃线虫的新种地位。通过比较舟山假滑刃线虫和贾氏球滑刃线虫 Tylaphelenchus jiaae 的系统进化关系、形态学以及生物学特性等，揭示 T. jiaae 应为假滑刃线虫属 Pseudaphelenchus 成员。

5. 滑刃线虫属 Aphelenchoides 五个未知种的描述

从中国和其他国家地区的木样和土样中分离到 6 个滑刃线虫属 Aphelenchoides 群体，根据形态学特征初步鉴定为 5 个不同种，分别是 Aphelenchoides sp. 12Asp、 Aphelenchoides sp. 14Asp、Aphelenchoides sp. 18Asp、Aphelenchoides sp. AspX 和 Aphelenchoides sp. Asp25。这 5 个未知种雌虫尾端均有单个尾尖突，属滑刃线虫属第 2 组成员，但是该组成员的鉴定存在较大的难度。首次发现 Aphelenchoides sp. 18Asp 的交合刺末端有分叉这一特征。基于 rDNA 18S 和 28S D2-D3 序列构建发育树分析了这 5 个未知种的系统进化关系，丰富了该属的分类学信息。

6. 滑刃科线虫头区形态特征及其系统进化关系

对滑刃线虫属 6 个群体的头区形态结构进行了扫描电子显微镜观察，经过与滑刃线虫科 11 属 78 个种的头区特征进行比较分析，根据口孔、内唇感受器、外唇感受器、头感器、侧器开口以及头区的环纹特征对滑刃科线虫的头区进行归类，获得 40 种头区类型。基于滑刃科线虫群体的 rDNA 18S 和 28S D2-D3 共计 404 个序列构建了合一系统进化树，该进化树揭示出滑刃线虫科各属成员分别聚类在 5 个主要的进化支中。头区类型

可以有效区分贝西滑刃线虫 Aphelenchoides besseyi 和福建滑刃线虫 A. fujianensis，然而头区类型与系统进化之间并不完全关联，关于滑刃科线虫头区形态结构特征与系统进化的关系有待进一步深入研究。

关键词：滑刃线虫科；滑刃线虫属；希尔滑刃线虫属；假滑刃线虫属；形态学；形态

测计；新种；头区；扫描电子显微镜；系统进化；合一分析

Chapter I General Introduction

The family Aphelenchoididae Skarbilovich, 1947 is a large group of nematodes which have a thin stylet with indistinctive basal swellings and a well developed metacorpus. The nematodes in this group have adapted to a wide range of ecological relationships, such as plant-parasitism, predation, fungi-feeding, obligate insect-parasitism, and associated with insects (Nickle, 1970a; Hunt, 1993, 2008). Due to economical importance, some plant-parasites in the family, i.e., Aphelenchoides besseyi, A. ritzemabosi, A. fragariae, Bursaphelenchus xylophilus and B. cocophilus, have received appropriate attention. Especially, the pine wilt nematode B. xylophilus (Steiner & Buhrer, 1934) Nickle, 1970 and other members of genus Bursaphelenchus are relatively well morphological and molecular characterized (Braasch et al., 2009; Ye et al., 2007). However, most of the genera in Aphelenchoididae are still unclear, especially due to the limited quality and missing molecular data in older descriptions, but also because of the high level of convergence of charetristics revealed in recent studies (Kanzaki & Giblin-Davies, 2012).

Classification

The systemic status of aphelench nematodes has gone through a series of changes. The majority of classification schemes classify aphelenchs as suborder Aphelenchina Geraert 1966 under the order Tylenchida (Andrássy, 1976; Luc et al., 1987; Maggenti et al., 1987; Nickle & Hooper, 1991; Maggenti, 1991). However, Siddiqi (1980, 2000) and Hunt (1993) considered aphelenchs as a separate order, i.e., Aphelenchida, and debated on significant morphological differences (i.e., amphid position, origin of stylet shaft and knob, dorsal pharyngeal gland orifice, metacorpus, anus structure, sperm, genital papillae or bursal limb and spicules) and certain biological aspects to warrant ordinal status of aphelenchs. Substantial changes in the higher taxa have occurred,and as a result of molecular methodologies the aphelenchs are no longer being recognized at either ordinal or subordinal rank (De Ley & Blaxter, 2002, 2004). Although Andrássy (2007) adhered aphelenchs to the ordinal rank, while Hunt (2008) retained the systemic status of “aphelenchs” nematodes as a single superfamily, Aphelenchoidea Fuchs, 1937.

The superfamily Aphelenchoidea is composed of two phylogenetically not closely related families, Aphelenchidae Fuchs, 1937 and Aphelenchoididae Skarbilovich,

1947 as shown in the phylogenetic tree of Tylenchomorpha (Fig. 1-1). The family Aphelenchidae clustered with other tylenchids and is positioned as a basal clade of tylenchids (Bert et al., 2008; Holterman et al., 2006, 2009; van Megen et al., 2009). On the other hand, the family Aphelenchoididae clustered with the superfamily Panagrolaimoidea Thorne, 1937 and apparently apart from the tylenchid clade. However, a phylogenetic analyses based on mitochondrial genomes indicated a monophyletic independent status of Aphelenchidae and Aphelenchoididae (Kim et al., 2015). Morphologically, Aphelenchidae can be distinguished from Aphelenchoididae and the major differences are listed below.

1. Spicules slender, gubernaculum well-developed, pharynx with distinctive isthmus, nerve ring surrounding pharynx, lateral lines number with 6 or more ...... Aphelenchidae

2. Spicules robust, rosethorn-shaped or derived there froms, gubernaculum absent, or if present, composed of small structure around distal tip of spicules, pharynx without distinctive isthmus, nerve ring surrounding pharynx and intestine, lateral lines number with 6 or less ...... Aphelenchoididae

The family Aphelenchidae comprises 2 subfamilies Aphelenchinae Fuchs, 1937 which is monotypic with the genus Aphelenchus Bastian, 1865 and the subfamily Paraphelenchinae Goodey, 1951 (Goodey, 1960) which is also monotypic with the genus Paraphelenchus Micoletzky, 1922. The family Aphelenchoididae contains 6 subfamilies in both authoritative systematic schemes in Hunt (1993, 2008). Several revisions of Aphelenchoididae, including the re-establishment of Devibursaphelenchus (Braasch, 2009), the discovery of the new genus Pseudaphelenchus (Kanzaki et al., 2009a), the record of several fossil genera of Aphelenchoididae (Poinar, 2011), the revision of Aphelenchoididae by Kanzaki & Giblin-Davies (2012), and the separation of Schistonchus s.l. into Schistonchus s.s., Ficophagus and Martininema by Davies et al. (2015), resulted in a new systematic scheme of family Aphelenchoididae which is listed below (following Kanzaki & Giblin-Davies 2012, with including recent adaptattions).

Family Aphelenchoididae Skarbilovich, 1947 Subfamily Aphelenchoidinae Skarbilovich, 1947 Aphelenchoides Fischer, 1894 Anomyctus Allen, 1940 Ficophagus Davies & Bartholomaeus, 2015 Laimaphelenchus Fuchs, 1937 Martininema Davies & Bartholomaeus, 2015 Punchaulus De Ley & Coomans, 1996 Robustodorus Andrássy, 2007 Ruehmaphelenchus Goodey, 1963 Schistonchus sensu strico Cobb, 1927 Sheraphelenchus Nickle, 1970 Tylaphelenchus Rühm, 1956 Pseudaphelenchus Kanzaki & Giblin-Davis, 2009 Subfamily Seinurinae Husain & Khan, 1967 Seinura Fuchs, 1931 Aprutides Scognamiglio, Talame & S’Jacob, 1970 Papuaphelenchus Andrássy, 1973 Subfamily Ektaphelenchinae Paramonov, 1964 Ektaphelenchus Fuchs, 1937 Cryptaphelenchus Fuchs, 1937 Devibursaphelenchus Kakuliya, 1967 Ektaphelenchoides Baujard, 1984 Subfamily Acugutturinae Hunt, 1980 Acugutturus Hunt, 1980 Noctuidonema Remillet & Silvain, 1988 Vampyronema Hunt, 1993 Subfamily Parasitaphelenchinae Ruhm, 1956 Parasitaphelenchus Fuchs, 1930 Bursaphelenchus Fuchs, 1937 Subfamily Entaphelenchinae Nickle, 1970 Entaphelenchus Wachek, 1955 Peraphelenchus Wachek, 1955 Praecocilenchus Poinar, 1969

Roveaphelenchus Nickle, 1970

Genera incertae sedis: Berntsenus1 Massey, 1974* Chalcidonema2 Poinar, 2011 Cretaciaphelenchoides2 Poinar, 2011 Oligoaphelenchoides2 Poinar, 1977 Palaeoaphelenchoides2 Poinar, 2011 Setonema2 Poinar, 2011

Note:

1. Berntsenus was treated as a member of subfamily Ektaphelenchinae (Hunt, 1993; 2008). However, this genus has a conspicuous anus and rectum which doesn't match the subfamily definition and no molecular data of this genus are available. Therefore, Kanzaki & Giblin-Davies (2012) have put this genus into incertae sedis.

2．For the fossil genera from amber materials both of morphological and molecular data are not available.

Ecology and plant-parasitism of Aphelenchoididae

The species in Aphelenchoididae are predominantly mycetophagous, can be found worldwide in soils, decaying plant tissues, and are especially abundant on the tree bark and in the tunnels of wood-boring beetles. The biodiversity and ecological behavior in Aphelenchoididae is very complex (Fig. 1-2). Some members of Aphelenchoididae have close relationships with insects by ectoparasitism, for example all life stages of Acugutturus and Noctuidonema are only found in its host American cockroach, Periplaneta americana and various noctuids, respectively (Hunt, 1980, Anderson & Laumond, 1992). Endoparasitic Aphelenchoididae, for example the Entaphelenchinae nematodes have a special stage in the insect hemocoel of various beetles, i.e., a mature parasitic female with large and swollen bodies (Remillet & Silvain, 1988; Hunt, 1993). Predators are also the important members of Aphelenchoididae as they rapidly thrust their stylets to penetrate the cuticle of their prey and inject immobilizing secretions before ingesting the body contents, e.g. Devibursaphelenchus (Gu et al., 2010a), Seinura (Hechler, 1963; Loof & Hooper,

1993), and Ektaphelenchus (Gu et al., 2013a, b). Some species of Aphelenchoididae are reported to be marine, e.g. Aphelenchoides gynotylurus was found in a salt water pool on the Bay of Bengal and A. marinus was found from blades of turtle grass (Thalassia sp.) in Biscayne Bay, Florida (Timm & Franklin, 1969).

Fig. 1-1. Schematic overview of the phylum Nematoda with division into 12 major clades derived from SSU rDNA sequence data (based on Holterman et al., 2006 and van Megen et al., 2009). Trophic ecologies are visualized by icons, and only the feeding preferences relevant in the context of this review are given. To illustrate the terms onchiostyle, odontostyle, and stomatostylet, examples are used from Clades 1, 2, and 12, respectively. This figure is adapted from Quist et al. (2015).

Plant-parasitism is polyphyletic in the phylum Nematoda, this acquired lifestyle evolved independently in each of three major clades (Blaxter et al. 1998; Baldwin et al. 2004), and among three traditional nematode orders (suborders), Dorylaimida Pearse 1942, Triplonchida Cobb 1920, and Rhabditida Chitwood 1933 (Fig. 1-1). Within these orders, 12 clades were established by van Megen et al. (2009), two important plant parasitic genera Bursaphelenchus and Aphelenchoides belong to clade 10 and other major plant parasitic nematodes belong to clade 12. The most studied effectors to date, the plant cell wall-degrading enzymes (CWDEs) with two distinct glycosyl hydrolase families (GH16, GH5 and GH45), which have been thought to be acquired from bacteria or fungi via the horizontal gene transfer (HGT) mechanism may show another view to demonstrate the different origin of plant parasitism (Jones et al., 2005; Haegeman et al., 2011; Haegeman et al., 2012; Palomares-Rius et al., 2014; Danchin et al., 2016; Wu et al., 2016) ,which was already proposed by Siddiqi 1983 (Fig.1-2).

Fig. 1-2. Evolution of plant-parasitism in nematodes. Knobbed ends represent predators. Numbered evolutionary lines refer to the following taxa. 1. Diphtherophoroidea; 2. Trichodoroidea; 3. Dorylaimoidea (partim); 4. Nygolaimoidea, Aporcelaimidae, Discolaimidae; 5. Tylencholaimoidea; 6. Longidoroidea, Dorylaimoidea (partim); 7. Tylenchoidea; 8. Dolichodoridae, Hoplolaimidae,

Criconematoidea; 9. Anguinidae; 10. Neotylenchoidea; 11. Sphaerularioidea, Iotonchioidea (Now moved to Hexatylina); 12. Aphelenchidae, Aphelenchoididae; 13. Aphelenchoididae (Adapted from Siddiqi, 1983).

The genera Bursaphelenchus and Aphelenchoides have received more scrutiny than any other members in Aphelenchoididae, although Schistonchus can also feed on plant cells within the Ficus carica fruit (Vovlas et al., 1992). Two classical Bursaphelenchus species responsable for plant diseases of major economic importance are the red ring nematode B. cocophilus and the pine wood nematode B. xylophilus. B. cocophilus is an obligate phytoparasite of coconut and oil plam trees, and is vectored by palm weevils. B. xylophilus is vectored by longhorn beetles belonging to the genus Monochamus, to pine trees where it reproduces in the vascular system with the associated blue-stain fungi which could be food sources for propagates of B. xylophilus. A newly identified species B. sycophilus is an obligate phytoparasite on fig syconia, Ficus variegate and is vectored by fig wasps (Kanzaki et al., 2014a). Another strategy taken by Aphelenchoides nematodes (colloquially known as “ bud and leaf nematodes”), is a facultative instead of obligate plant-parasitic nematode. Aphelenchoides besseyi, A. fragariae and A. ritzemabosi have 91, 620 and 321 plant species as their host, respectively (Sánchez-Monge et al., 2015), and are considered as facultative plant-parasites, e.g. A. besseyi and A. fragariae readily feed on fungi and A. ritzemabosi can reproduce on fungal mycelium as well (Hooper & Cowland, 1986). However the species do not induce re-differentiation of plant cells. Thus, the parasitic behavior in Aphelenchoides is far less complex and supposedly more primitive, which is potentially interesting to study the evolution of plant-parasitism (Jones et al., 2013).

General morphology

BODY HABITUS

The members of Aphelenchoididae are mostly slender and vermiform with body length varying from 0.2 to 2 mm and a little less than 1 mm in an average adult nematodes. Some ectoparasites and endoparasites of insects genera are swollen and the endoparasites are particularly obese reaching length of 3 mm or more. The body habitus of Aphelenchoididae is generally slightly curved when heat-killed, but the

8 male of Peraphelenchus is exceptional as it is cork-screw-shaped (Fig. 1-3).

Fig. 1-3. General morphology of Aphelenchoididae. A: Cryptaphelenchus iranicus, female and male; B: Ektaphelenchoides spondylis, female and male; C: Bursaphelenchus rufipennis, female and male; D: Noctuidonema guyanense, female; E: Vampyronema daptria, female; F: Praecocilenchus rhaphidophorus, female; G: Peraphelenchus necrophori, male; H: Roveaphelenchus jonesi, male. Drawings adapted from Esmaeili et al. (2016a); Kanzaki et al. (2009b); Kanzaki et al. (2008); Remillet & Silvain (1988); Anderson & Laumond (1992); Poinar Jr (1969); Wachek (1955). (Scale bars = 100 μm)

HEAD REGION

The cephalic sclerotization is usually weak. The cephalic region of Aphelenchoididae is typically hexaradiate with cephalic sectors of almost equal size.. The oral aperture is circular to oval in shape and is surrounded by a circle of six inner labial papillae as shown in Hooper & Clark, 1980 (Fig. 1-4). There are six bumps external to the oral aperture that may represent the outer labial papillae and four cephalic papillae in the submedian position. The amphidial apertures are small and pore-like and located dorsosublaterally on the lateral cephalic sectors (Hooper & Clark, 1980). After a detailed comparison of the head region from different Aphelenchoididae genera, we found that there are many differences in the cephalic pattern among the genera and species. These findings are addressed in detail in Chapter VII.

Fig. 1-4. Basic cephalic plate pattern in Aphelenchoididae (Hooper & Clark, 1980).

CUTICLE AND LATERAL REGION

The cuticle of Aphelenchoididae is generally thin and weak annulated. The lateral field is usually marked by various number of incisures by 0 (an unidentified Entaphelenchiae species ) to 6. However, some genera have multiple incisures (more than 6), e.g. Ficophagus zealandicus (Zhao et al., 2015). The incisures are known to be useful for species diagnosis andare congruent with phylogenetic clades of Bursaphelenchus (Ye et al., 2007; Braasch et al., 2009). However, in Aphelenchoides,

10 the lateral lines show variation, e.g. in A. nechaleos and A. paranechaleos (Hooper & Ibrahim, 1994), and the possibilities how lateral lines can be (wrongly) interpretated in Aphelenchoides was given in Sánchez-Monge (2016) (Fig. 1-5).

Fig. 1-5. Isometric projections of various Aphelenchoides lateral sides and corresponding possible interpretational morphology of the incisures under light microscopy (Sánchez-Monge 2016).

STYLET

The stylet in Aphelenchoididae is generally present (with the length from 10-20 μm), except for dauer stages of some species in Parasitaphelenchinae. The stylet is exceptionally long in Noctuidonema, up to 185 μm, and also in other genera of Acugutturinae the stylet can be 50-70 μm long (Fig. 6).

The stylet of Aphelenchoididae is normally thin and composed of two parts, i.e., conus and shaft, but it can also be robust in Schistonchus s.l. and Robustodorus (Fig. 1-6). The conus is usually shorter than the shaft but longer in Acugutturinae and some species of Schistonchus s.l. The stylet lumen is generally narrow, especially in Cryptaphelenchus. The base of stylet could have weak swellings or is small with conspicuous knobs, e.g. the basal knobs of the stylet of Schistonchus s.l. are relatively large. Furthermore, Robustodorus has typical massive knobs with a guiding apparatus in the conus. Exceptionally, some insect ectoparasites (e.g. Entaphelenchinae) and predators (e.g. Seinurinae and most genera of Ektaphelenchinae) have a stylet with wide lumen, and basal swelling or knobs entirely absent.

Fig. 1-6. Variation in stylet of Aphelenchoididae. Drawings modified after Adeldoost et al. (2016), Anderson & Laumond (1992), Davies et al. (2015), Esmaeili et al. (2016), Hooper & Cooke (1967), Hunt (1980), Nickle (1970a), Poinar (1969), Pourjam & Bert (2006), Remillet & Silvain (1988), Ryss et al. (2013), and Wang et al. (2013). (Scale bars = 20 μm)

PHARYNX

The pharynx is the most distinctive character of Aphelenchoididae and comprises a a procorpus and a metacorpus (median bulb). The procorpus is a uniformly narrow and cylindrical tube which is connected to the stylet and metacorpus. In Acugutturinae, which has a long stylet, the procorpus is retracted into a zigzag shape. The metacorpus forms a well-developed oval to rounded rectangular and muscular median bulb with prominent crescentic valve plates at the central or slight posterior part. Although the shape of the median bulb and its structure are quite similar in all genera, variation exist in some genera; well-developed pharyngeal glands are visible into the median bulb in Entaphelenchinae and the anterior one-third part of the median bulb is glandular in many genera of Ektaphelenchinae and Seinurinae (Fig. 1-7). Three pharyngeal gland cells have openings in the pharynx lumen inside the metacorpus, with the dorsal gland orifice anterior to the valve plate and the other two subventral gland orifices located at posterior to the plate. The pharyngo-intestinal junction is situated immediately posterior to the metacorpus and the isthmus is absent. The pharyngeal glands form a dorsally overlapping lobe on intestine.

Fig. 1-7. Metacorpus of Aphelenchoididae. A: Anterior body part in most Aphelenchoididae (Bursaphelenchus rufipennis adapted from Kanzaki et al., 2008); B: Metacorpus in most Aphelenchoididae (B. firmae adapted from Kanzaki et al., 2012); C: Bursaphelenchus; D: Aphelenchoides; E: Glandular part in 1/3 anterior of metacorpus in Ektaphelenchinae and Seinurinae (Seinura tenuicaudata adapted from Hechler, 1963); F: Metacorpus in Seinurinae; G: Metacorpus in Ektaphelenchinae; H: Pharyngeal glands into metacorpus in Entaphelenchinae (Kanzaki et al., 2013a); I: Metacorpus in Peraphelenchus; J: Metacorpus in Praecocilenchus (P. rhaphidophorus adapted from Poinar Jr 1969). (Scale bars = 10 μm)

FEMALE REPRODUCTIVE SYSTEM The gonoduct structure of Aphelenchoidea’s females (excluding mature parasitic females of Entaphelenchinae) is composed of an ovary, oviduct, spermatheca, crustaformeria, uterus or uterine sac, vagina/vulva and post-uterine sac (Fig. 1-8). However, each organ may not be clear enough to be distinctly visualized in microscopic observations without extraction from female body. The cellular architecture of the female gonduct in aphelenchs was revealed by Geraert (1973) and Bert et al. (2008) (Fig. 8).

Fig. 1-8. General morphology of female reproductive tract. A, B: Female reproductive tract in Aphelenchoides and Bursaphelenchus (Adapted from Kanzaki, 2006; Kanzaki et al., 2014b); C, D: The cellular architecture of oviduct, spermatheca and distal part of uterus in A. fragariae and Laimaphelenchus penardi (Adapted from Bert et al., 2008). (cr: crustaformeria; pus: post-uterine sac; ova: ovary; ovi: oviduct; sp: spermatheca; vu: vulva). (Scale bars = 10 μm)

The ovary is single and outstretched, reflexed in some species, oocytes are arranged as single or double rows at the anterior part, single to multiple rows in the middle part, and large cells arranged into a single row in the posterior part. The oviduct is short with flattened cells in most species (genera), e.g. the oviduct of Aphelenchoides trivialis, A. fragariae and Laimaphelenchus penardi is consisted of two flattened cells

(Fig. 1-8 C, D). The spermatheca is not clearly separated from other parts or is missing but conspicuous in Bursaphelenchus, generally as a branch by overlapping oviduct. Sometimes, the elongate tubular spermatheca comprises three groups of five to eight spermatheca cells, which surrounds a distinct lumen (Fig. 1-8). The crustaformeria is offset from uterus in most genera. The distal part of the uterus wall is formed by longitudinally arranged elongated cells; however, a special bilateral uterine gland complex is present in Vampyronema. The post-uterine sac is usually present and obvious, but very short or absent in several species (genera). Vulva is a simple slit in ventral view. A vulva flap is present in some species of Bursaphelenchus and Laimaphelenchus (Fig. 1-9).

Fig. 1-9. Variation of female vulval flap structure. Without the vulval flap, ventral view (A) and lateral view (B). Short vulval flap (= side flap) in ventral view (C) and lateral views (D-F). Long vulval flap in ventral view (G) and lateral view (H). Arrows in ventral view indicate the focal plane of corresponding lateral views. The a, b and c in C correspond to D, E and F, respectively. (Adapted from Kanzaki, 2008)

MALE COPULATORY SYSTEM

The male genital system in Aphelenchoididae is monorchic and composed of a testis and vas deferens. The testis has large and rounded sperm cells in single or multiple rows. The vas deferens posterior end is fused with the rectum to form a narrow cloacal tube. Spicules in Aphelenchoididae are separated and rosethorn-shaped or derivation of this shape with condylus (apex), capitulum, rostrum, calomus, lamina and cucullus (Fig. 1-10)

. Fig. 1-10. Morphology of male spicules. A, B: Mode of spicules in Aphelenchoididae (Bursaphelenchus xylophilus and B. seani, drawings adapted from Mamiya & Kiyohara, 1972, Giblin-Davis & Kaya, 1983); C: Bursaphelenchus obeche (Gu et al., 2007); D: B. debrae (Hazir et al., 2007); E: Schistonchus caprifici (Vovlas et al., 1992); F: Aphelenchoides besseyi (Jesus et al., 2016); G: A. paraxui (Esmaeili et al., 2017a); H: Pseudaphelenchus vindai (Kanzaki et al., 2010); I: Ektaphelenchoides winteri (Hooper, 1995); J: Ruehmaphelenchus formosanus (Kanzaki et al., 2013b); K: Peraphelenchus orientalis (Kanzaki et al., 2013a); L: Noctuidonema guyanense (Anderson & Laumond, 1990). (Scale bars = 10 μm)

The variation of spicules shapes in Aphelenchoididae is remarkable. In Noctuidonema, the shaft of the spicule is broad with a complicated arrangement of sheaths and sclerotized parts (Fig. 1-10 L). In the “Africanus” group of Bursaphelenchus, the spicule is straight with weakly developed and not-offset condylus and rostrum,

16 dorsally cross-striped in the middle part (Fig. 1-10 C). The gubernaculum is absent in most Aphelenchoididae, only a small and indistinct gubernaculum-like structure is present in Schistonchus and Acugutturus. Bursa is usually absent, but a true peloderan bursa supported by three pairs of limb-like papillae is present in Pseudaphelenchus, and a terminal flap of cuticle (bursa flap) in Parasitaphelenchinae (except for Bursaphelenchus kiyoharai) and Devibursaphelenchus (Fig. 1-11).

Three pairs of subventral caudal papillae (P2: one pre-cloacal, i.e., at level of cloacal aperture or slightly anterior, P3: one post-cloacal, and P4: one near the tail tip) is considered as an important character in this family and is usually present in most genera. A single precloacal midventral papilla (P1) could be observed in some species of Bursaphelenchus and Ruehmaphelenchus.

Fig. 1-11. Male tail. A-B: Ventral view of male tail in Bursaphelenchus (adapted from Kanzaki et al., 2007, and modified after Braasch et al., 2007); C: Ventral view of male tail in Pseudaphelenchus (Adapted from Kanzaki et al., 2010); D, E: Lateral view of male tail in Bursaphelenchus and Aphelenchoides (Adapted from Kanzaki et al., 2007, and Čermák et al., 2012). (P1: ventral single papilla; P2-P4: subventral paired papillae). (Scale bars = 10 μm)

TAIL

The tails of both sexes in Aphelenchoididae are generally short and conoid with variable shapes. It is an important character for identification both at genera and species level in this family. In the subfamily Seinurinae, the tail of female is long and elongate and the tail tip of both female and male rapidly tapers to filiform to a finely

17 point terminus in Seinura, a clavate terminus in Aprutides and a finely rounded terminus in Papuaphelenchus. A spike-like projection on the tail terminus is present in males of Ektaphelenchoides and Sheraphelenchus. An offset tail terminal peg with two to four clearly pedunculated tubercles is present in most Laimaphelenchus. While the tail shape of Aphelenchoides shows the most complicated inter-specific variation, which is important for intra-generic grouping in Aphelenchoides (Shahina, 1996; Hockland, 2001), for example no mucro (A. microstylus and A. obtusus), a star shape terminus (A. besseyi and A. goodeyi), a single terminus with mucro (A. subtenuis), a single terminal with tiny nodular protuberances mucro (A. xui and A. paraxui), a bifurcate terminal (A. bicaudatus). However, a considerably intraspecific variation is also possible.

Fig. 1-12. Variation of female tail. A: Aprutides martuccii; B: Ruehmaphelenchus martinii; C: Laimaphelenchus cocuccii; D: Bursaphelenchus xylophilus; E: Aphelenchoides obtusus; F: A. subtenuis; G: A. fragariae; H: A. xui; I: A. besseyi; J: A. ritzemabosi; K: A. bicaudatus; L: Seinura citri. Original drawings modified after from Doucet (1992), Fortuner (1970), Hechler &Taylor (1965), Mamiya & Kiyohara (1972), Rühm (1955), Scognamiglio et al. (1970), Siddiqi (1974; 1975; 1976), Thorne & Malek (1968), and Wang et al. (2013). (Scale bars = 20 μm)

Molecular identification and phylogeny of Aphelenchoididae

Molecular tools have been relatively recently applied to characterise and identify nematodes and other organisms. Molecular phylogenetic frameworks have allowed to have better insight in morphological patterns. Already in 2000, Siddiqi forwarded that the use of DNA and mitochondrial sequences, protein characterization by immunoelectrophoresis and monoclonal antibodies in identification of species, subspecies and races/pathotypes is gaining momentum. Among them, three loci of the ribosomal DNA (rDNA) region, near full length SSU (small subunit) or 18S rDNA gene, internal transcribed spacer (ITS) including ITS1, ITS2 and 5.8S 18S rDNA gene and D2-D3 expansion segments of LSU (large subunit) or 28S rDNA gene (Fig. 1-13.) have been routinely used as molecular markers for both free-living nematodes (Bhadury et al., 2006; Derycke et al., 2010; Floyd et al., 2002) and plant-parasitic nematodes (Holterman et al., 2009; Janssen et al., 2016; Lesufi et al., 2015; Powers, 2004). Within these three loci, 18S is due his conserved nature most suitable for analysis on higher taxonomic levels (Woese et al., 1990; Smit et al., 2007). The 28S D2-D3 is also relatively conserved and variable at the same time which make it suitable for species differentiation among closely-related genera, within a genus or at lower taxonomic ranking (Kanzaki & Giblin-Davis, 2012), for example it worked well to distinguish foliar Aphelenchoides nematodes at species level (Rybarczyk-Mydłowska et al., 2012; Sánchez-Monge et al., 2017). The ITS loci has the highest mutation rate of tandem duplication resulting in the highest variation among the three loci (Smit et al., 2007), which makes it suitable to distinguish closely related species or different population within a species.

Fig. 1-13. Structure of the ribosomal DNA gene family in nematodes. Coding regions of the 18S small subunit (SSU), 5.8S and 28S large subunit (LSU), non-coding regions of internal transcribed spacers (ITS1 and ITS2)

Besides the rDNA region, the mitochondrial genes play an important role in molecular markers for species identification, especially the mitochondrial cytochrome oxidase subunit I (COI) gene, which is used as the standard barcode for almost all animal groups (Hebert et al., 2003). However, using COI as molecular marker was only carried out in limited genera of Aphelenchoididae. Including Bursaphelenchus

(Kanzaki & Futai, 2002a, 2002b; Ye et al., 2007), Aphelenchoides (Sánchez-Monge et al., 2017) and Schistonchus s.l. species (De Luca et al., 2010), providing COI sequences for both species identification as for phylogenetic analysis. Furthermore, the analysis based on mitochondrial genomes of Aphelenchoididae has been used to supply new information of phylogenetic relationship among nematodes (Sultana et al., 2013, Sun et al., 2014).

Current molecular phylogenetic relationships of Aphelenchoididae inferred from 18S and 28S D2-D3 of rDNA show similar topologies (Kanzaki & Giblin-Davis, 2012; Ryss et al., 2013; Kanzaki et al., 2014c), four major clades are forwarded (Kanzaki & Giblin-Davis, 2012).

Clade 1 comprises of only one genus i.e., Pseudaphelenchus, which has an intermediate morphology between Aphelenchoididae and Aphelenchidae in respect to the aphelenchoidid-like pharynx and spicule but aphelenchid-like true male bursa with bursal limb-like genital papillae. However, based on the molecular phylogenetic analyses, Aphelenchus and Pseudaphelenchus are not inferred to be sisters (Kanzaki et al., 2009a, 2010, 2014).

Clade 2 is divided into two distinctive subclades 2a and 2b, which confirmed the paraphyly of Aphelenchoides as , Ficophagus, Laimaphelenchus, Martininema, and Schistonchus are embedded within Aphelenchoides. Ficophagus and Martininema are two monophyletic clades positioned within clade 2a, while Schistonchus s. s. was placed in clade 2b as a monophyletic clade. Aphelenchoides and Laimaphelenchus are placed into clade 2a and 2b (Wang et al., 2013; Esmaeili et al., 2016b; Sánchez-Monge et al., 2017). Especially Aphelenchoides is very difficult to identify due to low inter-specific and high intra-specific morphological variability, and most species are not well described or with old descriptions, without discriminating molecular data (Hockland 2001). This has already led to several misidentification, i.e., the misidentification of A. ritzemabosi and A. fragariae mentioned in Sánchez-Monge et al. (2017). Both Aphelenchoides and Laimaphelenchus should be re-examined and the generic system should be reconstructed based upon the newly collected detailed molecular and morphology data (Kanzaki & Giblin-Davis, 2012; Sánchez-Monge et al., 2017).

The Clade 3 composed of several diverse genera of Aphelenchoididae, e.g., predator

20 genera Anomyctus, Ektaphelenchus, Devibursaphelenchus and Seinura, and insect ectoparasitic genera Cryptaphelenchus, Ektaphelenchoides and Noctuidonema with complex relationships.

The Clade 4 consisted of Bursaphelenchus spp. and derived genera from Bursaphelenchus, Sheraphelenchus and Ruehmaphelenchus. The latter two are clades imbedded within Bursaphelenchus making this genus paraphyletic. Two remarkable species, B. abruptus and A. stammeri, show inconsistent phylogenetic relationships in 18S and 28S D2-D3 rees. According to the putative plesiomorphic single P1 ventral genital papilla in these two species, the B. abruptus and A. stammeri clade is suggested to reside within Bursaphelenchus spp. by Braasch (1998) and Urek et al. (2007).

OBEJECTIVES AND THESIS OUTLINE

Objectives

The aim of this thesis is to contribute and update several aspects of the family Aphelenchoididae by including more populations from different habitats, extracting detailed morphology, expanding the available molecular data by adding new DNA sequences and finding phylogentic relationships and its implemenations for the classification of Aphelenchoididae.

Outline

Chapter I: general introduction

Chapter II: Aphelenchoides stellatus n. sp. was isolated from packaging wood from Japan imported to Ningbo Port, China and described based on morphological and molecular data. It was characterised by a star-like tail terminus in male and female.

Chapter III: Aphelenchoides rotundicaudatus n. sp. was isolated from packaging wood from South Korea and described based on morphological and molecular data. It was characterised by the anteriorly located excretory pore and a cylindrical tail with a broadly rounded terminus in male and female.

Chapter IV: Two Sheraphelenchus populations were isolated from pine wood packaging from Italy and onion bulbs from South Korea at Ningbo Port, China, respectively. Based on morphology and molecular sequencing, the former one was described as S. parabrevigulonis n. sp and the other was identified as S. sucus. The phylogenetic trees of Sheraphelenchus were made to reveal the relationship of this genus within Aphelenchoididae.

Chapter V: Pseudaphelenchus zhoushanensis n. sp. was isolated from a dead Pinus thunbergii tree at Zhoushan Islands, China and described based on morphological and molecular data. The combination of the molecular phylogenetic analyses, morphology and biology of P. zhoushanensis n. sp. and Tylaphelenchus jiaae indicates that T. jiaae is a member of Pseudaphelenchus to which it is herein transferred as P. jiaae n. comb. (= T. jiaae).

Chapter VI: Six Aphelenchoides populations, which belong to the Group 2 according to the category of Aphelenchoides species by the female tail shapes having a single mucro, were isolated from different habitats from the different countries. The large number of species in Group 2 makes the diagnosis of five unknown species difficult. All these six populations were characterized as five different and putative new species based on morphological and molecular characters.

Chapter VII: The diversity of head patterns characters in Aphelenchoididae is analyzed. A comparison of head region characters of 78 species from 11 Aphelenchoididae genera revealed 40 types of head regions. A concatenated phylogeny analyses by combining 404 sequences of 18S and D2-D3 28S rDNA revealed five major clades of Aphelenchoididae. Plotting the morphological data on this tree revealed that the head region types are useful to distinguish several closely related species, however a congruence with the phylogenetic clades was not observed

Chapter II

Description of Aphelenchoides stellatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from Japan

Chapter published as:

Fang, Y. 1, Gu, J.2, Wang, X.1 and Li, H.1 (2014). Description of Aphelenchoides stellatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from Japan. Nematology 16, 621-630. 1Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R. China. 2Technical Centre, Ningbo Entry-Exit Inspection and Quarantine Bureau, 9 Mayuan Road, Ningbo 315012, Zhejiang, P.R. China.

Abstract

Aphelenchoides stellatus n. sp. is described and figured. The new species was isolated from packaging wood from Japan imported to Ningbo harbour, China. The new species has a body length of 485-533 μm (males) and 547-699 μm (females). The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 9-11 μm long and has small basal swellings. The excretory pore is located posterior to the nerve ring. Spicules smoothly curved, rose-thorn shaped. Apex and rostrum round, only slightly offset, dorsal limb 19-21 μm long. Male tail bearing six (2 + 2 + 2) caudal papillae. The female spermatheca is axial and oblong with round sperm present in multiple rows. Both male and female tail pegs have 3-4 processes, appearing star-like under SEM. The new species belongs to the Group 3 category of Aphelenchoides species. Phylogenetic analyses based on full length ITS and 28S D2-D3 region of rDNA confirmed its morphological status as a new species.

Keywords – molecular, morphology, morphometrics, new species, phylogeny, SEM, taxonomy.

Introduction

Since 1991, China has implemented quarantine regulations for coniferous packaging wood imported with shipped commodities in order to prevent the spread of the pine wood nematode, Bursaphelenchus xylophilus, and other pests. Almost all packaging wood imported through Ningbo harbour has been inspected and sampled since 1997. From these inspections, many Bursaphelenchus spp. have been detected (Gu et al., 2006). However, some species belonging to other genera of Aphelenchoididae have also been detected from imported wood packaging materials (Gu et al., 2010b, 2013a, b; Wang et al., 2013). Some Aphelenchoides species also have been reported from pine woods in China (Zhuo et al., 2010), including A. besseyi Christie, 1942, which is listed as a regulated pest and characterised by a star-like female tail tip (EPPO, 2004). In August 2008, one new species of Aphelenchoides isolated from packaging wood from Japan was intercepted, which is described in this paper.

Materials and methods

CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS

Sawn samples taken from packaging wood were cut into small pieces and nematodes were extracted by the modified Baermann funnel technique for 24 h. Populations were multiplied and maintained on Botryotinia fuckeliana. Measurements were performed on permanent slides fixed by heat-killed nematodes fixed with FA 4:1 and ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). The light micrographs were made using a Zeiss microscope equipped with a Zeiss AxioCam MRm CCD camera. The SEM micrographs were taken with a Hitachi S3400 scanning electron microscope.

MOLECULAR STUDIES

DNA samples of nematodes were prepared according to Li et al. (2008). Two sets of primers (synthesized by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the full length ITS and the partial 28S D2/D3 regions of rDNA, respectively. The ITS1/2 region was amplified with the forward primer F194 (5’-CGT AAC AAG GTA GCT GTA G-3’) (Ferris et al., 1993) and the reverse primer 5368r (5’-TTT CAC TCG CCG TTA CTA AGG-3’) (Vrain, 1993). The D2/D3 domain region of 28S was amplified with the forward primer D2A (5’-ACA AGT ACC GTG

AGG GAA AGT TG-3’) and the reverse primer D3B (5’-TCG GAA GGA ACC AGC TAC TA-3’) (De Ley et al., 1999). PCR conditions were as described by Li et al. (2008). PCR products were separated on 2% agarose gels and visualised by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Invitrogen.

The full length ITS and partial 28S D2/D3 sequences of A. stellatus n. sp. were aligned by Clustal W and compared with those of the other Aphelenchoides species available in GenBank using the BLAST homology search program. The tree topology for each gene was carried out separately using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The best fitted model of DNA evolution was obtained using Modeltest v.3.7 (Posada & Crandall, 1998). Phylogenetic analyses then used the Akaike-supported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC. The trees were confirmed by running the chain for 1 × 106 generations and setting the ‘burnin’ at 1000. The Markov Chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using 50% majority rule (Larget & Simon, 1999). The consensus trees were selected to represent the phylogenetic relationships with branch length and support level and visualised using TreeGraph2 (Stöver & Müller, 2010).

Results

Aphelenchoides stellatus* n. sp.

(Figs 2-1, 2-2, 2-3)

* Specific epithet formed from the Latin word stella, and referring to the star-like tail terminus in both male and female.

MEASUREMENTS

See Table 2-1.

DESCRIPTION

Male

Body slender, cylindrical, J-shaped when heat-relaxed. Cuticle weakly annulated, lateral field with four incisures (i.e., three ridges). Lip region offset by slight

28 constriction. Stylet delicate with small basal swellings. Median bulb strongly developed, almost oval, 12.7 ± 0.7 (11.7- 14.0) μm long × 8.6 ± 0.2 (8.3-8.9) μm broad, with more or less central valve plates. Nerve ring situated just posterior to median bulb. Pharyngeal glands slender, ca 3-4 body diam. long, overlapping intestine dorsally. Excretory pore located posterior to never ring. Hemizonid faint. Testis outstretched, ca 248 ± 17.5 (227- 280) μm long. Lips of cloacal opening protruding slightly. Spicules smoothly curved, rose-thorn-shaped, 14-17 μm long. Apex and rostrum rounded, only slightly offset, dorsal limb 19-21 μm long. Gubernaculum absent. Tail conical, terminus bearing a peg with 3-4 processes, appearing star-like with SEM. Three pairs of subventral caudal papillae present: first pair located just posterior to cloacal aperture, second pair in mid-tail region, third pair just anterior to tail end. Bursa absent.

Female

Body slightly ventrally arcuate when heat-relaxed. Cuticle and lip region similar to male. Ovary outstretched, developing oocytes in single row. Spermatheca present, 2-3 body diam. long, containing round sperm in multiple rows. Quadricolumella obscure. Post-uterine sac well developed, extending for ca one-third of vulva to anus distance, sperm often present. Vagina directed slightly anteriad. Vulva transverse, with slightly raised lips, vulval flap absent. Rectum and anus visible. Tail elongate conoid, slightly ventrally arcuate, terminus armed with a tetramucronate process, giving it a star-like appearance under SEM.

Table 2-1. Morphometrics of Aphelenchoides stellatus n. sp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n – 15 15 L 496 510 ± 16.7 (485-533) 638 ± 51.0 (547-699) a 38.2 38.1 ± 1.9 (35.8-41.0) 42.3 ± 1.8 (39.9-44.8) b 8.1 7.7 ± 0.4 (6.7-8.2) 9.1 ± 0.8 (7.8-10.1) b' 4.1 4.3 ± 0.3 (3.8-4.8) 4.4 ± 0.4 (3.7-4.8) c 15.5 16.9 ± 0.8 (15.5-17.9) 17.6±2.3 (14.0-20.9) c' 2.9 2.8 ± 0.1 (2.7-2.9) 3.7 ±0.4 (3.2-4.5) V or T 38.7 47.3 ± 3.6 (38.5-54.2) 69.2 ± 1.3 (65.5-70.2) Body diam. 13 13.4 ± 0.5 (12.9-14.6) 15.1 ± 1.7 (12.3-17.5) Stylet length 9.9 10.0 ± 0.2 (9.6-10.3) 10.1 ± 0.5 (9.1-11.0) Median bulb length 11.7 12.7 ± 0.7 (11.7-14.0) 13.1 ± 0.6 (12.1-14.0) Median bulb diam. 8.3 8.6 ± 0.2 (8.3-8.9) 8.8 ± 0.6 (8.3-10.0) Median bulb 1.4 1.5 ± 0.1 (1.4-1.6) 1.5 ± 0.1 (1.3-1.7) length/diam. Excretory pore 67 67 ± 2.3 (64-72) 72 ± 3.3 (66-75) position Spicule (chord) 16 15.8 ± 0.6 (14.4-16.8) - Spicule (curved - 15.3 15.4 ± 1.1 (13.5-16.9) median line) Spicule (dorsal limb) 20.1 19.6 ± 0.5 (18.7-20.6) - Spicule (ventral 11.8 10.2 ± 1.0 (8.7-11.8) - limb) Ovary or testis 192 238 ± 17.5 (192-280) 219 ± 34.0 (147-269) length Post-uterine sac - - 49.9 ± 3.6 (45.0-57.0) Vulva to anus - - 158 ± 12.6 (133-170) distance Post-uterine sac - - length/ 32.4 ± 3.6 (28.0-38.3) vulva to anus (%) Mucro - - 1.5 ± 0.1 (1.3-1.6)

Fig. 2-1. Aphelenchoides stellatus n. sp. A: Entire female; B: Entire male; C: Anterior body; D: Female vulval region; E-G: Tail tips; H: Female tail; I: Male tail; J: Spicule. (Scale bars = 10 μm.)

Fig. 2-2. Light photomicrographs of Aphelenchoides stellatus n. sp. A: Female; B: Male; C: Lateral lines; D: Vulval region; E: Head region; F, G: Female tail terminus; H-J: Male tail. (Scale bars = 10 μm.)

Fig. 2-3. Scanning electronic microscopy of Aphelenchoides stellatus n. sp. A: En face view of female; B: Vulva; C: Lateral region; D-F: Tail terminus.

TYPE HABITAT AND LOCALITY

Isolated from packaging wood of Pinus sp. Exported from Japan and inspected at Ningbo, P.R. China. Nematodes were cultured on a lawn of Botryotinia fuckeliana growing on PDA.

TYPE MATERIAL

Holotype male and three paratype females (slide no N-02) deposited in the nematode collection of Nanjing Agricultural University, P.R. China, seven paratype males and 11 paratype females (slide no T494a and T494b) in the Canadian National Collection of Nematodes, Ottawa, ON, Canada, 25 paratype males and 31 paratype females (slide no. 8503-1 to 8503-11) ) in the nematode collection of Ningbo Entry-Exit Inspection and Quarantine Bureau.

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides stellatus n. sp. is characterised by body lengths of 485-533 μm (male) and 547-699 μm (female). The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 9-11 μm long and has small basal swellings. The excretory pore is located posterior to the nerve ring. The spicules are smoothly curved, rosethorn shaped, and 14-17 μm long with the apex and rostrum rounded, only slightly offset, and the dorsal limb 19-21 μm long. The male tail bears six (2 + 2 + 2) caudal papillae. The spermatheca is axial and oblong and contains round sperm in multiple rows. Both male and female terminus bear a peg with 3-4 processes, appearing star-like under SEM. According to the categorization codes of EPPO (2004), A. stellatus n. sp. is defined as A1-B1/2-C1-D1/2-E1-F3.

According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 3 which is defined as having tail terminus with “tetramucronate spine or star-shape structure”. Based on lateral line number, star-shaped tail terminus and stylet length, it is close to six species from Group 3, including A. besseyi Christie, 1942, A. fujianensis Zhuo, Cui, Ye, Luo, Wang, Hu & Liao, 2010, A. goodeyi Siddiqi & Franklin, 1967, A. hylurgi Massey, 1974 (see Bird et al., 1989), A. panaxofolia Liu, Wu, Duan & Liu, 1999, and A. siddiqii Fortuner, 1970.

Aphelenchoides stellatus n. sp. differs from A. besseyi by the excretory pore position (posterior to nerve ring vs near anterior edge of nerve ring). It differs from A. fujianensis by the shorter body length of 0.64 (0.56-0.70) vs 0.86 (0.80-0.94) mm, excretory pore position (posterior to nerve ring vs near anterior edge of nerve ring) and the shorter spicule length of 15.4 (13.5-16.9) vs 26 (24-30) μm. It differs from A. goodeyi by the female ratio a (39.9-44.8 vs 29-39) and the presence of males. It differs from A. hylurgi by the female ratio a (40-45 vs 22-35) and the post-uterine sac length (ca three vs one body diam.). The new species differs from A. panaxofolia in the

34 length of the dorsal limb of the spicule (18.7-20.6 vs 14.3-16.3 μm) and the number of male caudal papillae (3 vs 2 pairs). It differs from A. siddiqii by the female ratio a (40-45 vs 27-39) and ratio c’ (3.7 vs 2.6), the female tail shape (elongate conoid vs subcylindroid) and the postuterine sac length (ca 3 vs 0.5-1.0 body diam.).

Aphelenchoides stellatus n. sp. originated from pine wood, a habitat where three Aphelenchoides species with a star-shaped tail terminus have been reported, including A. besseyi, A. fujianensis and A. menthae Lisetskaya, 1971 (Zhuo et al., 2010). The new species differs from A. menthae mainly by the number of lateral lines (4 vs 2).

MOLECULAR CHARACTERISATION AND PHYLOGENY

The sequences of full length ITS (1069 bp, isolate 8503, GenBank accession number FJ768943) and partial region 28S D2/D3 region (814 bp, KF638651) of A. stellatus n. sp. were determined. Alignment of the sequences with ClustalW resulted in datasets of 1309 characters for ITS1/2, and 984 characters for 28S D2/D3. Phylogenetic relationships among the isolates for each dataset were determined using Bayesian inference (BI) with Aphelenchus avenae as outgroup. The 50% majority rule consensus phylogenetic trees were generated from ITS1/2 and 28S D2/D3 dataset alignment by BI analysis under the TrN + I + G model and GTR + I + G model, respectively. The ITS1/2 tree (Fig. 2-4) showed A. stellatus n. sp. to be closest to A. paradalianensis (HQ454505), but with only 63% sequence similarity. However, A. stellatus n. sp. differs from A. paradalianensis in the tail terminus of both female and male (star tipped with 3-4 processes vs a short trunk with two fine processes).

The 28S D2/D3 tree (Fig. 2-5.) revealed that A. stellatus n. sp. clustered with two unknown species of Aphelenchoides and other species of Aphelenchoides, Laimaphelenchus and Schistonchus. The new species shared sequences similarity with two unknown species by 79.3% (isolate CA22, DQ328682) and 79.6% (K1 WY-2008, EU287597), respectively. However, the two unknown spices shared a high similarity (99.6%), which may provide indicate that they are conspecific. Molecular profiles and phylogenetic analyses clearly support the proposal of A. stellatus n. sp. as a new species.

Fig. 2-4. Phylogenetic relationships of Aphelenchoides stellatus n. sp. and aphelenchid nematodes based on full length ITS. The 10 001st Bayesian tree inferred from ITS1/2 ITS under TrN + I + G model (ln L = 17 620.0195; freqA = 0.2484; freqC = 0.2034; freqG = 0.2426; freqT = 0.3055; R(a) = 1.0000; R(b) = 2.3567; R(c) = 1.0000; R(d) = 1.0000; R(e) = 3.6572; R(f) = 1.0000; Pinvar = 0.0558; Shape = 1.0675). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Fig. 2-5. Phylogenetic relationships of Aphelenchoides stellatus n. sp. and aphelenchid nematodes based on partial 28S. The 10 001st Bayesian tree inferred from 28S D2/D3 under GTR + I + G model (ln L = 14 183.3984; freqA = 0.1752; freqC = 0.1893; freqG = 0.3342; freqT = 0.3013; R(a) = 1.0410; R(b) = 3.8444; R(c) = 1.1803; R(d) = 0.6390; R(e) = 4.9652; R(f) = 1.0000; Pinvar = 0.2360; Shape = 0.7926). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Discussion

The FAO Guidelines for Regulating Wood Packaging Material in International Trade (ISPM 15) have been followed by many countries since 2003. All exported package materials should be properly heat- or methyl bromide-treated and officially marked in line with ISPM 15 (Wang et al., 2013). However, in recent years, quite a number of living nematodes have been repeatedly detected in Ningbo port from imported packaging wood bearing ISPM marks. Most of the nematode isolates were primarily identified as Aphelenchoides spp. and Bursaphelenchus spp. and kept as cultures or permanent slides for further identification. One isolate from South Africa was identified as Aphelenchoides xui (Wang et al., 2013). The description of the new species enriched our knowledge on Aphelenchoides species having a star-like tail terminus, especially for those wood-dwelling species.

Due to the inadequate morphological descriptions of many of the earlier published Aphelenchoides species, the precise diagnosis of a new species of Aphelenchoides can be extremely difficult. It is likely that many of the older-described species will never be reliably reidentified, but tying molecular data to current standard descriptions offers a way forward from this impasse. As molecular diagnostics are now well developed, they can provide useful information to assist morphological identification. Although the phylogenetic analyses using the Bayesian method revealed A. stellatus n. sp. has a close relationship with A. paradalianensis, based on ITS sequences, and with an Aphelenchoides sp. Based on 28S rDNA sequences, the morphological characters strongly supported its status as a new species. The topology of the trees also revealed that some species currently in the genera Aphelenchoides, Laimaphelenchus and Schistonchus have a close relationship, which may infer that these taxa could share a most recent common ancestor and that these clades have not speciated very far. Rybarczyk-Mydłowska et al. (2012), based on SSU analyses using the Bayesian method, also concluded that Aphelenchoides species did not constitute a monophyletic group. In general, new species/descriptions of Aphelenchoides can only be reliably diagnosed by using a combination of molecular and traditional techniques.

According to Hunt (2008) there are about 153 described species of Aphelenchoides. Since then, about ten new species have been described in detail. As more aphelenchid sequences become available in GenBank, the entire Aphelenchoides group is likely to be revised on the basis of morphological characters observed with the light microscopic and SEM and combined with molecular taxonomic evidence.

Chapter III

Description of Aphelenchoides rotundicaudatus n. sp. (Nematoda: Aphelenchoididae) found in

packaging wood from South Korea

Chapter published as:

Fang, Y. 1, Wang, X.1, Gu, J.2 and Li, H.1 (2014). Description of Aphelenchoides rotundicaudatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from South Korea. Nematology 16, 751-760.

1Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R. China. 2Technical Centre, Ningbo Entry-Exit Inspection and Quarantine Bureau, 9 Mayuan Road, Ningbo 315012, Zhejiang, P.R. China.

Abstract

Aphelenchoides rotundicaudatus n. sp. was isolated from packaging wood from South Korea imported to Ningbo harbour, China. Specimens were collected directly from wood samples using the Baermann method. Multiplication on Botryotinia fuckeliana failed. The new species has a body length of 364-509 μm (males) and 371-493 μm (females). The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 8-9 μm long and has small basal swellings. The excretory pore is located ca one body diam. anterior to median bulb. Spicules are small (10-13 μm), apex and rostrum rounded and only slight offset. The male tail has six (2 + 2 + 2) caudal papillae. Both male and female tails are cylindrical with a broadly rounded terminus, often with a small region of thickened cuticle or a blunt peg ca 1 μm long, particularly in the female. The new species belongs to the Group 1 category of Aphelenchoides species. Phylogenetic analyses based on full length ITS, partial LSU and SSU of rDNA confirmed its status as a new species.

Keywords: molecular, morphology, morphometrics, new species, phylogeny, SEM, taxonomy.

Introduction

The FAO Guidelines for Regulating Wood Packaging Material in International Trade (International Standards for Phytosanitary Measures (ISPM) No. 15, 2009) have been followed by many countries since 2003. All exported package materials should be compulsorily heat- or methyl bromide-treated and officially marked in order to eliminate the pine wood nematode, Bursaphelenchus xylophilus, and other pests (ISPM). However, in recent years, quite a number of living nematodes have been repeatedly detected in Ningbo port from imported packaging wood bearing ISPM marks. Most of the nematode isolates were primarily identified as Aphelenchoides spp. and Bursaphelenchus spp. and kept as cultures or permanent slides for further identification. In July 2008, a new species of Aphelenchoides isolated from packaging wood exported from South Korea was intercepted at Ningbo harbour and is described herein.

Materials and methods

CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS

Sawn samples collected from the wood packaging materials were sliced into small chips ca 1 cm wide and nematodes were extracted by the modified Baermann funnel technique for 24 h. Adults isolated from samples were prepared for permanent mounts by heat-killing and fixing with FA 4:1 followed by ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). Drawings, measurements and light micrographs of nematodes on permanent slides were done with the aid of Zeiss microscope equipped with a Zeiss AxioCam MRm CCD camera.

MOLECULAR STUDIES

DNA templates of nematodes were prepared according to Li et al. (2008). Three sets of primers (synthesised by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify parts of the SSU, ITS1/2 and LSU regions of rDNA, respectively. The SSU region was amplified with the forward primer 5’-AAA GAT TAA GCC ATG CAT G-3’ and the reverse primer 5’-AGT CAA ATT AAG CCG CAG-3’ (Wang et al., 2013). The ITS1/2 region was amplified with the forward primer F194 (5’-CGT AAC AAG GTA GCT GTA G-3’) (Ferris et al., 1993) and the reverse primer 5368r (5’-TTT CAC TCG CCG TTA CTA AGG-3’) (Vrain, 1993). The D2-D3 domain region of LSU

41 was amplified with the forward primer D2A (5’-ACA AGT ACC GTG AGG GAA AGT TG-3’) and the reverse primer D3B (5’-TCG GAA GGA ACC AGC TAC TA-3’) (De Ley et al., 1999). PCR conditions were as described by Li et al. (2008). PCR products were separated on 2% agarose gels and checked by staining with ethidium bromide. PCR products demonstrated with high quality were purified for cloning and sequencing by Invitrogen.

The full length ITS, partial LSU and SSU sequences of A. rotundicaudatus n. sp. were compared with those of other Aphelenchoides species deposited in GenBank using the BLAST homology search tool. The tree topology for each gene was determined separately using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The base substitution model was evaluated using Modeltest v.3.7 (Posada & Crandall, 1998). Phylogenetic analyses then implemented the Akaike-supported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC. The trees were confirmed by running the programs as described by Fang et al. (2014). The phylogenetic relationships were presented by consensus trees selected by branch length and support level and visualised using TreeGraph2 (Stöver & Müller, 2010).

Results

Aphelenchoides rotundicaudatus* n. sp.

(Figs 3-1, 3-2)

* Etymology: the specific epithet referring to the broadly rounded tail terminus in both male and female.

MEASUREMENTS

See Table 3-1.

DESCRIPTION

Male

Body ventrally C-shaped when heat-relaxed. Body slender, cylindrical, ventrally arcuate especially posterior to spicules when heat-relaxed. Cuticle weakly annulated, lateral field with four incisures (i.e., three ridges). Lip region rounded, offset, ca 2.4 μm high and 4.9 μm broad. Stylet with small basal swellings. Median bulb strongly

42 developed, oval, 10.1 ± 1.3 (7.4-12.5) μm long × 7.8 ± 1.1 (6.3-10.2) μm broad, with more or less central valve plates. Nerve ring situated just posterior to median bulb. Pharyngeal gland lobe slender, ca 4-5 body diam. long, overlapping intestine dorsally. Excretory pore located ca one body diam. anterior to median bulb. Hemizonid faint, situated ca one body diam. posterior to median bulb. Testis prodelphic, outstretched, developing spermatocytes arranged in single row. Lips of cloacal opening protruding slightly. Spicules smoothly arcuate, rose-thorn shaped, 10-13 μm long. Apex and rostrum rounded, only slightly offset, dorsal limb 14-18 μm long. Gubernaculum absent. Tail cylindrical, terminus usually broad rounded, occasionally bearing a short and blunt cuticular thickening or peg (present in 46% of males). Three pairs of subventral caudal papillae present: first pair located just posterior to cloacal aperture, second pair in mid-tail region, third pair just anterior to tail end. Bursa absent.

Female

Body ventrally arcuate or C-shaped when heat relaxed. Cuticle and lip region similar to male. Ovary outstretched, developing oocytes in single row. Spermatheca present, containing oval sperms in a single row. Quadricolumella obscure. Vagina directed anteriad. Vulval lips slightly protruding, without flap. Post-uterine sac well developed, extending for ca two-thirds of vulva to anus distance, sperm often present. Rectum and anus visible. Tail cylindrical, slightly ventrally arcuate, terminus broadly rounded with a very small cuticular thickening or bearing a blunt peg (present in 97% of females) ca 1 μm long.

Table 3-1. Morphometrics of Aphelenchoides rotundicaudatus n. sp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n – 15 15 L 388 433 ± 46.9 (364-509) 433 ± 42.7 (371-493) a 38 39.1 ± 2.7 (34.6-43.6) 39.0 ± 4.5 (31.9-46.5) b 7.8 7.5 ± 0.6 (6.5-8.7) 7.9 ± 1.0 (6.7-9.5) b' 4.0 4.1 ± 0.4 (3.4-4.7) 4.4 ± 0.5 (3.8-5.2) c 17.6 16.7 ± 1.6 (13.9-19.9) 16.3 ± 2.3 (13.4-21.8) c' 2.8 3.0 ± 0.3 (2.5-3.4) 3.6 ± 0.3 (2.9-4.0) V or T 48.2 45.1 ± 5.8 (37.4-54.9) 68.5 ± 2.2 (65.8-75.1) Body diam. 10.2 11.1 ± 1.5 (8.9-14.7) 11.1 ± 0.7 (10.1-12.) Lip height 2 2.4 ± 0.3 (1.9-3.2) 2.3 ± 0.2 (1.9-2.7) Lip diam. 4.9 4.9 ± 0.8 (2.2-6.1) 5.0 ± 0.2 (4.6-5.4) Stylet length 8.5 12.7 ± 0.7 (11.7-14.0) 13.1 ± 0.6 (12.1-14.0) Median bulb length 9.2 10.1 ± 1.3 (7.4-12.5) 10.1 ± 0.5 (9.4-11.0) Median bulb diam. 6.7 7.8 ± 1.1 (6.3-10.2) 7.5 ± 0.4 (6.8-8.0) Median bulb 1.4 1.3 ± 0.2 (1.0-1.6) 1.4 ± 0.1 (1.2-1.5) length/diam. Excretory pore 28.4 ± 2.6 (24.7-33.7) 27 29.1 ± 3.6 (22.2-33.6) position Spicule (chord) 11 1.6 ± 0.8 (10.1-13.0) - Spicule (curved - 10.8 10.9 ± 0.7 (9.9-12.4) median line) Spicule (dorsal limb) 14.1 14.9 ± 1.1 (13.6-17.5) - Spicule (ventral limb) 8.7 9.2 ± 0.7 (8.1-10.6) -- Ovary or testis length 187 185 ± 37.1 (151-275) 130 ± 22.5 (95-171) Post-uterine sac - - 68 ± 11.1 (52-89) Vulva to anus distance - - 111 ± 12.6 (89-131) Post-uterine sac - - length/ 63 ± 8.8 (47.1-78) vulva to anus (%) Tail length 22 26 ± 2.9 (21-32) 27 ± 2.2 (21-30) Anal body diam. 8 8.8 ± 1.2 (7.9-11.6) 7.5 ± 0.6 (6.3-8.5)

Fig. 3-1. Aphelenchoides rotundicaudatus n. sp. A: Entire female; B: Entire male; C: Anterior body; D: vulval region; E, F: Female tails; G: Male tail; H: Spicule. (Scale bars = 10 μm.)

Fig. 3-2. Light photomicrographs of Aphelenchoides rotundicaudatus n. sp. A: Head region; B: Pharyngeal region; C: Vulval region; D: Lateral lines; E: Post-uterine sac; F-H: Female tails; I-L: Male tails. (Scale bars = 10 μm.)

TYPE HABITAT AND LOCALITY

Type specimens were isolated directly from imported packaging wood of Pinus sp. from South Korea. The nematode did not culture on Botryotinia fuckeliana.

TYPE MATERIAL

Holotype male, 16 paratype males and 13 paratype females (slide no. 23556-1 to 23556-8) are deposited in the nematode collection of Ningbo Entry-Exit Inspection and Quarantine Bureau, two paratype males and three paratype females (slide no. 9145) in the Canadian National Collection of Nematodes, Ottawa, ON, Canada, and two paratype males and three paratype females (slide no. N-03) in the nematode collection of Nanjing Agricultural University, Nanjing, P.R. China.

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides rotundicaudatus n. sp. is characterised by body lengths of 364-509 μm and 371-493 μm for male and female, respectively. The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 8-9 μm long and has small basal swellings. The excretory pore is located ca one body diam. anterior to the median bulb. The spicules are smoothly arcuate, rose-thorn shaped and 10-13 μm long with the apex and rostrum rounded, only slightly offset, and the dorsal limb 14-18 μm long. The male tail bears six (2 + 2 + 2) caudal papillae. The tail of both male and female is cylindrical with a broadly rounded terminus, often with a cuticular thickening or bearing a blunt peg ca 1 μm long, particularly in the female. According to the categorisation codes of OEPP/EPPO (2004), A. rotundicaudatus n. sp. Is defined as A5-B2-C4-D2-E1-F1.

According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 1 which is defined as having the female tail “simple without any outgrowth or mucronate structure”. Based on lateral line number and the stylet length, it is close to seven species from Group 1, including A. capsuloplanus (Haque, 1967) Andrássy, 1976, A. confusus Thorne & Malek, 1968, A. limberi, Steiner, 1936, A. obtusicaudatus Eroshenko, 1967, A. obtusus Thorne & Malek, 1968, A. orientalis Eroshenko, 1968 and A. parasubtenuis Shavrov, 1967.

The new species differs from all these species by excretory pore position (ca one body diam. anterior to the median bulb vs near or posterior to nerve ring). Furthermore, it is also distinguished from A. capsuloplanus by the spicule length (10-13 vs 15-17 μm),

47 the female ratio a (31.9-46.5 vs 24-30.2) and female tail terminus (rounded with a cuticular thickening or a blunt peg vs rounded); from A. confusus by the stylet length of 8.6 (8.0-9.0) vs 12 μm, and female tail terminus (rounded with a cuticular thickening or a blunt peg vs rounded) and the presence of the male; from A. limberi by the female body length of 433 (371-493) vs 740 (639-960) μm, the dorsal limb length of the spicule of 14.9 (13.6-17.5) vs 32.2 (30.0-35.2) μm and the male tail shape (cylindrical broadly rounded, often with a cuticular thickening or a blunt peg vs conical hamate mucro); from A. obtusicaudatus by the female body length (371-493 vs 570-580 μm), female tail shape (subcylindroid vs conoid) and the presence of the male; from A. obtusus by the post-uterine sac length (ca four vs two vulval diam. long), the female ratio a (31.9-46.5 vs 26.0), the female tail terminus (rounded, often with a cuticular thickening or a blunt peg vs rounded) and the male tail shape (subcylindroid vs conoid); from A. orientalis by the female tail shape (subcylindroid vs conoid), the female ratio a (31.9-46.5 vs 19.7-21.9) and presence of the male; from A. parasubtenuis by the female body length (371-493 vs 160-210 μm), the female ratio a (31.9-46.5 vs 21.3-25.0) and ratio b (6.7-9.5 vs 5.3-6.4), the female tail terminus (a broadly rounded terminus usually with a cuticular thickening or a blunt peg vs a simple mucro) and presence of the male.

MOLECULAR CHARACTERISATION AND PHYLOGENY

The sequences of full length ITS (747 bp, isolate 23556, GenBank accession number KF772860), partial LSU region (775 bp, KF772859) and partial SSU (888 bp, KF772858) of A. rotundicaudatus n. sp. were determined. Alignment of the sequences with Clustal W resulted in datasets of 1166 characters for ITS1/2, 862 characters for LSU and 980 characters for SSU. Phylogenetic relationships among the isolates were determined separately for each dataset using Bayesian inference (BI) with Aphelenchus avenae as outgroup. The 50% majority rule consensus phylogenetic trees were generated from ITS1/2 and LSU dataset alignment by BI analysis under the GTR + I + G model, and from SSU under TrN + I + G model.

The SSU tree (Fig. 3-3) revealed that A. rotundicaudatus n. sp. clustered with three species of Aphelenchoides and other species of Aphelenchoides, Laimaphelenchus and Schistonchus. The new species shared sequences similarity with A. blastophthorus Franklin, 1957 (isolate AchoBla, AY284644), A. fragariae (Ritzema Bos, 1890) Christie, 1932 (isolate AfBotCad, DQ901551) and A. saprophilus Franklin, 1952

(isolate 2140, FJ040408) by 89.0%, 89.3% and 89.3%, respectively. Morphologically, the new species differs from A. blastophthorus by the female tail shape (subcylindroid, broadly rounded with occasionally a cuticular thickening or a blunt peg vs conoid with single terminal mucro), the female body length (371-493 vs 680-900 μm), the female stylet length (8.1-9.1 vs 14-19 μm), the dorsal limb length of the spicule (13.6-17.5 vs 24-31 μm), and excretory pore position (ca one body diam. anterior to the median bulb vs approaching the nerve ring); from A. fragariae by the female tail shape (subcylindroid vs elongated conoid), the lateral line number (four vs two), excretory pore position (ca one body diam. anterior to the median bulb vs approaching or posterior to nerve ring) and dorsal limb length of the spicule (13.6-17.5 vs 24-31 μm); from A. saprophilus by the female tail shape (subcylindroid vs conoid), excretory pore position (ca one body diam. anterior to the median bulb vs approaching or anterior to nerve ring) and the dorsal limb length of the spicule (13.6-17.5 vs 23 μm).

The LSU tree (Fig. 3-4) also revealed that A. rotundicaudatus n. sp. clustered with some species of Aphelenchoides, Laimaphelenchus and Schistonchus and formed an independent clade. The ITS1/2 tree (Fig. 3-5) showed A. rotundicaudatus n. sp. to be closest to an unknown species of Aphelenchoides (isolate US6996, FJ768940) with 68.6% sequence similarity. Molecular profiles and phylogenetic analyses strongly supported the status of A. rotundicaudatus n. sp. as a new species.

Fig. 3-3. Phylogenetic relationships of Aphelenchoides rotundicaudatus n. sp. and aphelenchid nematodes based on partial SSU. The 10001st Bayesian tree inferred from SSU under TrN + I + G model (ln L = 10 464.9150; freqA = 0.2594; freqC = 0.2100; freqG = 0.2498; freqT = 0.2808; R(a) = 1.0000; R(b) = 2.3248; R(c) = 1.0000; R(d) = 1.0000; R(e) = 3.6440; R(f) = 1.0000; Pinvar = 0.2397; Shape = 0.4845). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Fig. 3-4. Phylogenetic relationships of Aphelenchoides rotundicaudatus n. sp. and aphelenchid nematodes based on partial LSU. The 10001st Bayesian tree inferred from LSU under GTR + I + G model (ln L = 14 085.4072; freqA = 0.1708; freqC = 0.1927; freqG = 0.3395; freqT = 0.2970; R(a) = 0.9551; R(b) = 3.4871; R(c) = 1.1526; R(d) = 0.5195; R(e) = 4.2875; R(f) = 1.0000; Pinvar = 0.2443; Shape = 0.7980). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Fig. 3-5. Phylogenetic relationships of Aphelenchoides rotundicaudatus n. sp. and aphelenchid nematodes based on full length ITS. The 10001st Bayesian tree inferred from ITS1/2 ITS under GTR + I + G model (ln L = 17 768.6641; freqA = 0.2398; freqC = 0.2056; freqG = 0.2508; freqT = 0.3038; R(a) = 1.1657; R(b) = 2.3255; R(c) = 1.1982; R(d) = 0.8997; R(e) = 3.0307; R(f) = 1.0000; Pinvar = 0.1071; Shape = 1.4309). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Discussion

Aphelenchoides rotundicaudatus n. sp. is characterised by both male and female having a broadly rounded tail terminus with occasionally a cuticular thickening or a blunt peg, and belongs to Aphelenchoides species sensu Shahina (1996) Group 1, which was defined as having a tail that was “simple without any outgrowth or mucronate structure”. However, in our observation of individuals of the new species, the proportion having the tail terminating in a blunt peg was 46% in the male and 97% in the female.Variation in tail terminus shape was also reported in the Group 1 species A. limberi by Hooper (1962) and Čermák et al. (2012), where the female has a truncate to smoothly rounded tail terminus occasionally bearing a mucro.

Many of the earlier published species of Aphelenchoides suffer from inadequate

52 morphological detail which makes it difficult to compare putative new species with older descriptions. Molecular diagnostics, however, can assist in accurate identification, although with the important proviso that most nominal Aphelenchoides species lack such information. Although the phylogenetic analyses using the Bayesian method indicated A. rotundicaudatus n. sp. was close to three Aphelenchoides species, A. blastophthorus, A. fragariae and A. saprophilus, based on SSU sequences, and to an Aphelenchoides sp., based on ITS sequences, the morphological characters of the female tail shape and terminus clearly distinguished the new species from the species mentioned above.

The phylogenetic analyses of aphelenchids indicated the close relationships of Aphelenchoides, Laimaphelenchus and Schistonchus based on SSU, LSU and ITS sequences (Rybarczyk-Mydłowska et al., 2012; Wang et al., 2013; Fang et al., 2014). The tree topology based on those sequences of Aphelenchoides did not constitute an independent monophyletic group, and this may infer that they shared a recent common ancestor. As more DNA sequences of aphelenchids are deposited in GenBank, the entire Aphelenchoides group is likely to be revised on the basis of a combination of morphological characters and molecular diagnostics.

Chapter IV

Description of Sheraphelenchus parabrevigulonis n. sp. (Nematoda: Aphelenchoididae) in pine wood packaging from Italy and redescription of S. sucus in onion bulbs from South Korea, isolated at Ningbo, China

Chapter published as:

Fang, Y. 1, Gu, J.2, Wang, X.1, Wang, J.2 and Li, H.1 (2015). Description of Sheraphelenchus parabrevigulonis n. sp. (Nematoda: Aphelenchoididae) in pine wood packaging from Italy and redescription of S. sucus in onion bulbs from South Korea, isolated at Ningbo, China. Nematology 17, 213-229.

Abstract

Two Sheraphelenchus populations were isolated from pine wood packaging from Italy and onion bulbs from South Korea at Ningbo Port, China, respectively. The former, on the basis of morphology and molecular sequencing, is described as S. parabrevigulonis n. sp., which is characterised by a stylet length of 11 μm, three lines in the lateral field, excretory pore at level with, or slightly posterior to, nerve ring, V = 93.1, and spicules with long condyles and membrane-like rostrum in a shallow triangular shape. The species from onion, also on the basis of morphology and sequence data, was identified as S. sucus, which is characterised by a stylet length of 13 μm (erroneously stated to be ca 6 μm in the type description), excretory pore at level of, or posterior to, median bulb, and V = 80.9. The phylogeny trees based on rDNA 18S, 28S D2-D3 and ITS1/2 sequences revealed that S. parabrevigulonis n. sp. and S. sucus clustered into a clear clade and grouped with Bursaphelenchus species. The study enriched the taxonomic information of Sheraphelenchus and confirmed its status as an independent genus.

Keywords – molecular, morphology, morphometrics, new species, phylogeny, taxonomy.

Introduction

Sheraphelenchus Nickle, 1970 belongs to the Aphelenchoidinae and is distinguished from other genera in the subfamily by the well-developed stylet without basal knobs or swellings, vulva posterior with a V value larger than 80, and tail forming a digitiform spike with a finely pointed terminus (Hunt, 1993). The genus includes three nominal species: S. brevigulonis (Massey & Hinds, 1970) Hunt, 1993, S. entomophagus Nickle, 1970 (the type) and S. sucus Kanzaki & Tanaka, 2013. To date, S. sucus is the only species with both a detailed morphological description and molecular data, Kanzaki & Tanaka (2013) revealing the first view of the phylogenetic status of this rare genus and an especially close relationship with Bursaphelenchus.

During inspections of imported wood packaging and plant materials at Ningbo Port, China, two Sheraphelenchus isolates were obtained. According to the morphological and molecular characters, one isolate obtained from pine wood packaging from Italy in May 2012 was described as a new taxon, S. parabrevigulonis n. sp. Another isolate, obtained from onion bulbs from South Korea in September 2011, was identified on molecular data as S. sucus. However, due to its variation in stylet length from the original description, this S. sucus isolate was redescribed using morphological characterisations and phylogenetic analyses in order to provide a better interpretation of the genus and its relationships.

Materials and methods

CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS

Sawn wood packaging samples and onion bulbs were cut into small pieces and nematodes were extracted by a modified Baermann funnel technique for 24 h. Nematodes failed to culture on Botryotinia fuckeliana. Measurements were performed on permanent slides made by heat-killed nematodes fixed with FA 4:1 and ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). The light micrographs were made by a Zeiss Imager Z1 microscope equipped with a Zeiss AxioCam MRm CCD camera.

MOLECULAR STUDIES

DNA samples were prepared according to Li et al. (2008). Three sets of primers were used in the PCR analyses to amplify the partial 18S region, the 28S D2- D3 region

57 and the ITS1/2 region of rDNA. The 18S region was amplified with the forward primer K4f, 5’-ATG CAT GTC TAA GTG GAG TAT TA-3’, and the reverse primer K1r, 5’-TTC ACC TAC GGC TAC CTT GTT ACG ACT-3’ (Penas et al., 2006); the 28S D2-D3 region was amplified with the forward primer D2A and the reverse primer D3Br (De Ley et al., 1999), and the ITS1/2 region was amplified with the forward primer F194 (Ferris et al., 1993) and the reverse primer 5368r (Vrain, 1993). PCR conditions were as described by Li et al. (2008). PCR products were separated on 1.5% agarose gels and visualised by staining with Gelred (Biotium). PCR products of sufficiently high quality were purified and the cloning and sequencing was performed by Invitrogen.

The partial 18S, 28S D2-D3 and ITS1/2 sequences of S. parabrevigulonis n. sp. and S. sucus were compared with those of other Aphelenchoidea species available in GenBank using the BLAST homology search tool. The best-fit model of DNA evolution was obtained using Modeltest v.3.7 (Posada & Crandall, 1998). Phylogenetic analyses were carried out by implementing the Akaike-supported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC. The tree topology for each gene was determined separately using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The running programs of the trees were as described by Fang et al. (2014). The consensus trees were selected to represent the phylogenetic relationships with branch length and support level, and visualised using TreeGraph2 (Stöver & Müller, 2010).

Results

Sheraphelenchus parabrevigulonis* n. sp.

(Figs 4-1, 4-2, 4-3)

* Specific epithet formed from the Latin word para = beside or near, and the species epithet brevigulonis, thereby reflecting its close similarity to S. brevigulonis.

MEASUREMENTS

See Table 4-1.

DESCRIPTION

Male

Body slender, cylindrical, ventrally arcuate when heat-relaxed. Cuticle thin with fine annulations. Lateral field with three incisures. Lip region convex, distinctly offset from body. Stylet with small basal swellings. Procorpus cylindrical, ca 1.5 body diam. long. Metacorpus (median bulb) round, oval or pear-shaped, conspicuous valve plates situated almost centrally. Pharyngo-intestinal junction immediately posterior to end of metacorpus. Pharyngeal gland lobe ca 3-4 body diam. long, overlapping intestine dorsally. Nerve ring located ca 1-2 body diam. posterior to metacorpus. Excretory pore at level with or slightly posterior to nerve ring. Hemizonid at ca 1.5 body diam posterior to nerve ring. Gonad single, outstretched. Spermatocytes arranged in 2-5 rows for most of testis length and in a single row in posterior part. Tail region almost straight or slightly arcuate ventrally. Anterior half part of tail broadly conoid, then narrowing abruptly to form a digitiform spike tapering to pointed tip. Spike ca one anal body diam. long. Cloacal lips distinctly protruding. Spicules paired, separate. Condylus long with a rounded end, membrane-like rostrum shallow triangular in form. Dorsal limb slightly arcuate ventrally, tapering smoothly to a pointed distal end. Three pairs of subventral caudal papillae: one pair located just posterior to cloacal aperture, two pairs adjacent to one another and located just anterior to spike-like projection. Bursa absent.

Female

Body habitat similar to male when heat-killed. Cuticle and anterior body region similar to male. Gonad outstretched, occasionally reflexed. Developing oocytes present in multiple rows in anterior part of ovary. Several well-developed oocytes in a single row at posterior end. Oviduct tube-like, occasionally occupied by a well-developed oocyte. Spermatheca slightly irregular oval shape, sometimes filled with well-developed sperm. Crustaformeria not conspicuous. Uterus short, with thick wall, usually containing one or two embryonated eggs. Vagina slightly inclined anteriorly. Vulva located very posteriorly and close to anus, vulval flap lacking. Vulval lips protruding slightly. Post-uterine sac absent. Rectum and anus visible. Body abruptly narrowing posterior to vulval opening, tapering to a digitiform spike with a finely rounded or bluntly pointed terminus.

Table 4-1. Morphometrics of Sheraphelenchus parabrevigulonis n. sp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n – 20 20 L 535 545 ± 94 (387-790) 539 ± 51 (433-620) a 31.5 31.8 ± 5.8 (21.8-43.2) 27.4 ± 3.6 (23.0-35.1) b 10.4 11.3 ± 2.2 (9.0-18.6) 11.5 ± 1.5 (9.1-14.7) b’ 5.1 5.4 ± 0.7 (4.4-7.2) 5.3 ± 0.5 (4.6-6.1) c 22.2 25.5 ± 3.5 (20.4-34.0) 21.7 ± 3.8 (16.3-31.2) c’ 2.1 1.7 ± 0.2 (1.4-2.1) 2.2 ± 0.3 (1.7-3) T or V 65.6 66.5 ± 5.9 (57.0-77.2) 93.1 ± 0.6 (92.0-94.0) Max. body diam. 17.0 17.3 ± 2.6 (13.2-25.0) 19.9 ± 2.3 (15.4-23.5) Lip diam. 7.1 7.7 ± 0.7 (6.4-9.2) 7.7 ± 0.5 (7.0-8.5) Lip height 3.0 3.2 ± 0.4 (2.5-3.9) 3.4 ± 0.5 (2.7-4.1) Stylet length 12.4 11.5 ± 1.0 (10.2-12.6) 11.1 ± 0.7 (11.1-12.5) Median bulb diam. 9.6 10.0 ± 1.3 (7.8-12.1) 9.9 ± 0.8 (8.2-11.0) Median bulb length 13.5 14.3 ± 1.8 (10.6-16.9) 13.5 ± 1.3 (11.6-16.8) Median bulb length/diam. 1.4 1.5 ± 0.2 (1.3-1.9) 1.4 ± 0.1 (1.2-1.5) Excretory pore from anterior 81.1 73 ± 4.5 (64-81) 68 ± 7.8 (55-86) end Spicule (chord) 19.3 18.0 ± 2.69 (14.0-22.6) – Spicule (dorsal limb) 21.5 20.0 ± 3.9 (14.1-25.0) – Ovary or testis length 351.2 363 ± 68.7 (259-489) 362 ± 52.9 (290-449) Tail length 24.1 21.4 ± 2.7 (15.9-25.7) 25.6 ± 4.2 (19.9-36.4) Anal body diam. 11.4 12.6 ± 1.1 (11.1-15.4) 11.9 ± 1.8 (9.2-15.7) Hemizonid position 96.7 97 ± 5.9 (92-105) 89 ± 18.4 (68-101)

Fig. 4-1. Sheraphelenchus parabrevigulonis n. sp. A: Entire female; B: Entire male; C: Head region; D: Stylet; E: Posterior part of young female gonad; F: Lateral view of mature female tail; G: Lateral lines; H: Male tail; I: Ventral view of male tail. (Scale bars = 10 μm.)

Fig. 4-2. Light photomicrographs of Sheraphelenchus parabrevigulonis n. sp. male. A: Entire male; B-E: Head region; F, G: Caudal papillae indicated by arrows; H-O: Male tail. (Scale bars = 10 μm.)

Fig. 4-3. Light photomicrographs of Sheraphelenchus parabrevigulonis n. sp. female. A: Entire female; B: Head region; C: Lateral lines; D, E: Uterus with embryonated eggs; F-L: Lateral view of female tail; M: Ventral view of vulval region. (Scale bars = 10 μm.)

TYPE HABITAT AND LOCALITY

Isolated from packaging wood of Pinus sp. Exported from Italy and inspected at Ningbo Port, P.R. China. Nematodes failed to culture on a lawn of Botryotinia fuckeliana.

TYPE MATERIAL

Holotype male, 26 paratype males and 36 paratype females isolated from pine wood packaging from Italy were deposited in the nematode collection of Ningbo Entry-Exit Inspection and Quarantine Bureau (NBCIQ) (slides no. 1516-1 to 1516-15); four paratype males and five paratype females were deposited in Nanjing Agricultural University (NAU), P.R. China (slide nos N04-1 to N04-3); and three paratype females and two paratype males (slide nos T546a and T546b) were deposited in the Canadian National Collection of Nematodes (Ottawa, ON, Canada).

DIAGNOSIS AND RELATIONSHIPS

Sheraphelenchus parabrevigulonis n. sp. is characterised by body length of the male at 545 (387-790) μm and female at 539 (433-620) μm. The cuticle is weakly annulated with three incisures in the lateral field. The stylet is 11.5 (10.2-12.6) μm long with small basal swellings. The excretory pore is located at the same level as, or slightly posterior to, the nerve ring. The male tail has a 9.8 (8.0-10.6) μm long digitiform spike with a finely pointed terminus but lacks a bursa. Three pairs of subventral caudal papillae are present. The female vulva is located very posteriorly, V = 93 (92-94).

Sheraphelenchus parabrevigulonis n. sp. is close to S. brevigulonis, a poorly known species with a rather brief description, but apparently differs by the number of incisures in the lateral field (three vs not mentioned) and the number of male subventral caudal papillae (three pairs vs two pairs). Of course, it is possible that these differences merely reflect shortcomings in the original description but, until S. brevigulonis can be re-isolated from the type locality and sequenced, we feel it is more prudent to describe our isolate, which is complete with all morphological and molecular data, as a new taxon in order to provide a firmer platform from which to view the genus. It differs from S. entomophagus by the excretory pore position (slightly posterior to nerve ring vs anterior to nerve ring), V = 93.1 (92.0-94.0) vs 82.9 (77.8-88.1), male tail length (21.4 vs 38 μm), female tail length (25.6 vs 73 μm) and female c ratio of 21.7 (16.3-31.2) vs 8.7 (7.5-10.2). It differs from S. sucus by the

64 excretory pore position (slightly posterior to nerve ring vs anterior to nerve ring), V = 93.1 (92.0-94.0) vs 82.3 (80.3-85.1), male tail length of 21.4 (15.9-25.7) vs 49 (41-63) μm, female tail length of 25.6 (19.9-36.4) vs 82 (63-93) μm, male ratio c = 25.5 (20.4-34.0) vs 10.8 (8.5-13.8) and female ratio c = 21.7 (16.3-31.2) vs 7.6 (6.6-9.4).

S. sucus Kanzaki & Tanaka, 2013

(Figs 4-4, 4-5, 4-6)

MEASUREMENTS

See Table 4-2.

DESCRIPTION

Male

Body slightly ventrally arcuate when heat-relaxed. Cuticle marked by fine transverse striations, ca 1 μm wide. Lateral field ca 2 μm wide, with three equally spaced lines at mid-body. Lip region convex, not offset. Stylet ca 13 μm long, without basal knobs but with slight swellings. Procorpus cylindrical, very short. Median bulb round, oval or pear-shaped, conspicuous valve plates situated almost centrally. Pharyngo-intestinal junction immediately posterior to end of metacorpus. Pharyngeal gland lobe ca 3- 4 body diam. long, overlapping intestine dorsally. Nerve ring located ca 1-2 body diam. posterior to metacorpus. Excretory pore located at level of, or posterior to, metacorpus, ca 40-65 μm from anterior end. Hemizonid ca 84-108 μm from anterior end. Gonad single, outstretched, occupying ca three fifths of body length. Spermatocytes arranged in 2-5 rows for most of testis length and in a single row in posterior part. Tail region almost straight or slightly arcuate ventrally. Anterior one third part of tail broadly conoid, narrowing sharply to form a digitiform spike tapering to pointed tip. Spike ca three anal body diam. long. Cloacal lips distinctly protruding. Spicules paired, separate. Condylus long with a rounded end, rostrum low and triangular, membrane-like. Dorsal limb slightly arcuate ventrally, smoothly tapering to a pointed distal end. Three pairs of subventral caudal papillae present: one pair located immediately posterior to cloacal opening, two pairs adjacent to one another and just anterior to spike-like projection. Bursa absent.

Female

Body habitus similar to male when heat relaxed. Cuticle and anterior body region similar to male. Gonad outstretched, occupying ca three fifths of body length. Developing oocytes present in multiple rows in anterior part of ovary, several well-developed oocytes in a single row at posterior end. Oviduct tube-like, occasionally occupied by a well-developed oocyte. Spermatheca oval, sometimes filled with well-developed sperm. Crustaformeria formed of small, rounded cells, not conspicuous. Uterus short and wall thickened, usually containing one or two embryonated eggs. Vagina slightly inclined anteriorly. Vulva located very posteriorly, vulval flap lacking. Vulval lips slightly protruding. Post-uterine sac absent. Rectum and anus visible. Body gradually narrowing posterior to vulval opening, tapering smoothly to a conical tail with a finely rounded or bluntly pointed terminus.

Table 4-2. Morphometrics comparison of Sheraphelenchus sucus isolates. All measurements are in μm and in the form: mean ± s.d. (range).

Character S. sucus S. sucus isolate 10984 type isolate (Kanzaki & Tanaka, 2013) Male Female Male Female n 20 20 15 16 L 575 ± 70 597 ± 83 527 ± 41 623 ± 82 (494-751) (500-876) (483-601) (451-719) a 25.4 ± 1.9 24.2 ± 2.0 23.2 ± 2.5 22.8 ± 1.7 (22.6-30.4) (20.7-27.4) (19.4-27.8) (20.2-26.3) b 7.6 ± 0.8 8.2 ± 1.0 12.6 ± 0.9 14.5 ± 1.6 (6.7-9.7) (6.6-10.8) (11.5-14.4) (10.9-16.7) b’ 5.1 ± 0.6 5.4 ± 0.7 – – (4.3-6.5) (4.5-7.3) c 10.8 ± 1.0 7.5 ± 0.7 10.8 ± 1.4 7.6 ± 0.7 (9.3-12.8) (6.5-9.3) (8.5.-13.8) (6.6-9.4) c’ 4.1 ± 0.2 7.2 ± 0.7 3.9 ± 0.4 6.9 ± 0.7 (3.8-4.6) (5.9-8.8) (2.9-4.6) (5.8-8.3) T or V 59.9 ± 5.0 80.9 ± 0.9 56.6 ± 7.8 82.3 ± 1.2 (45.3-66.6) (79.0-82.8) (41.5-70.0) (80.3-85.1) Max. body diam. 22.7 ± 2.5 24.7 ± 2.6 23.0 ± 3.4 27.5 ± 4.6 (19.7-28.8) (20.6-33.2) (19.5-31.0) (17.5-33.0) Lip diam. 7.9 ± 0.5 8.1 ± 0.7 8.0 ± 0.5 8.5 ± 0.5

(6.9-8.8) (7.0-9.6) (7.5-9.0) (7.0-9.0) Lip height 3.1 ± 0.3 3.2 ± 0.3 3.5 ± 0.3 3.5 ± 0.3 (2.7-3.6) (2.8-4.0) (2.5-4.0) (3.0-4.5) Stylet length 13.5 ± 0.3 13.2 ± 0.4 6.5 ± 0.4** 6.5 ± 0.5** (12.8-14.0) (12.3-13.9) (5.5-7.0) (5.5-7.0) Median bulb diam. 11.8 ± 0.8 12.3 ± 1.0 12.0 ± 0.7 12.5 ± 1.3 (10.3-13.3) (10.7-13.8) (10.5-13.5) (10.5-14.5) Median bulb length 16.0 ± 0.7 17.0 ± 1.0 15.5 ± 0.7 16.5 ± 1.1 (14.7-17.4) (14.9-20.0) (14.0-17.0) (15.5-18.5) Median bulb 1.4 ± 0.1 1.4 ± 0.1 1.3 ± 0.1 1.3 ± 0.1 length/diam. (1.2-1.5) (1.3-1.5) (1.2-1.5) (1.1-1.5) Excretory pore from 52 ± 7.0 52 ± 7.4 6.5p ± 6.3* 9.5p ± 8.2* anterior end (40-65) (36-70) (6.5a-16.0p) (3.5a-22.0p) Spicule (chord) 20.4 ± 1.3 – 20.0 ± 1.2 – (18.5-23.2) (17.5-22.5) Spicule (dorsal limb) 22.1 ± 1.5 – – – (19.0-24.9) Ovary or testis length 343 ± 50.0 361 ± 67.2 300 ± 59 214 ± 65 (293-483) (233-555) (214-410) (109-314) Tail length 53 ± 3.5 80 ± 11.0 49 ± 6.1 82 ± 9.5 (48-59) (57-116) (41-63) (63-93) Anal body diam. 12.9 ± 0.8 11.1 ± 1.0 12.5 ± 0.8 12.0 ± 1.1 (11.8-14.5) (9.0-13.2) (12.0-14.5) (9.5-13.5) Hemizonid position 98 ± 6.4 98 ± 6.1 88 ± 4.0 93 ± 6.7 (84-108) (90-116) (83-100) (78-103) * The letters refer to whether the excretory pore is anterior (a) or posterior (p) to the end of the metacorpus. ** This is a very short measurement for the genus and could be the result of a misinterpretation by Kanzaki & Tanaka (2013).

Fig. 4-4. Sheraphelenchus sucus. A: Entire female; B: Entire male; C: Head region; D: Stylet; E: Lateral lines; F: Posterior part of young female gonad; G: Lateral view of mature female tail; H: Male tail; I: Ventral view of male tail. (Scale bars = 10 μm.)

Fig. 4-5. Light photomicrographs of Sheraphelenchus sucus male. A: Entire male; B, C: Head region, secretory-excretory pore indicated by arrow; D: Lateral lines; E, F: Caudal papillae (arrows); G, H: Male tail; I-N: Spicules. (Scale bars = 10 μm.)

Fig. 4-6. Light photomicrographs of Sheraphelenchus sucus female. A: Entire mature female; B: Entire young female; C: Vulval region; D: Female tail; E, F: Uterus without eggs; G-J: Uterus with embryonated eggs. (Scale bars = 10 μm.)

VOUCHER MATERIAL

Sixteen permanent slides of S. sucus isolated 10984 from onion bulbs from South Korea were deposited in NAU (slide nos R01-1 to R01-4) and NBCIQ (slide nos 10984-1 to 10984-12). Each slide contained several adults and juveniles.

DIAGNOSIS AND RELATIONSHIPS

The original specimens of S. sucus described by Kanzaki & Tanaka (2013) were isolated from a sap flow of Quercus serrata. Compared to morphological and morphometric data of the original isolate, our redescribed onion isolate of S. sucus was very similar to the type description, the major apparent differences being the male stylet length of 13.5 (12.8-14.0) vs 6.5 (5.5-7.0) μm, the female stylet length of 13.2 (12.3-13.9) vs 6.5 (5.5-7.0) μm, male b = 7.6 (6.7-9.7) vs 12.6 (11.5-14.4) and female b = 8.2 (6.6-10.8) vs 14.5 (10.9-16.7). The extremely short stylet length recorded by Kanzaki & Tanaka (2013) may be an underestimate, all other known records of the genus having a stylet about 12 μm long. Indeed, in Figure 3 of Kanzaki & Tanaka (2013), the LM may indicate that the conus and shaft have become detached from one another and slightly overlap, the procorpus being pushed well anteriad, possibly as a result of the fixation process. If this is the case, by measuring both putative parts, the total length of the stylet can be calculated to be in excess of 10 μm (the tip of the conus is not clearly visible could therefore be several microns longer).

The onion isolate of S. sucus differs from S. brevigulonis (Massey & Hinds, 1970) by the excretory pore position (anterior to nerve ring vs slightly posterior to nerve ring), V = 80.9 (79.0-82.8) vs 91 (91-93), male tail length of 53 (48-59) vs 23 μm, female tail length of 80 (57-116) vs 29 μm, male c ratio = 10.8 (9.3-12.8) vs 23.8 (16.4-27.7), and female c ratio = 7.5 (6.5-9.3) vs 22.0 (19.9-22.8). It differs from S. entomophagus (Nickle, 1970) by the male tail length of 53 (48-59) vs 38 μm and the number of incisures in lateral field (three vs not mentioned). It differs from S. parabrevigulonis n. sp. by the excretory pore position (anterior to nerve ring vs slightly posterior to nerve ring), V = 80.9 (79.0-82.8) vs 93.1 (92.0-94.0), male tail length of 53.3 (47.9-58.6) vs 21.4 (15.9-25.7) μm, female tail length of 80.0 (57.4-116.3) vs 25.6 (19.9-36.4) μm, male c ratio = 10.8 (9.3-12.8) vs 25.5 (20.4-34.0), and female c ratio = 7.5 (6.5-9.3) vs 21.7 (16.3-31.2).

MOLECULAR CHARACTERISATION AND PHYLOGENY

The sequences of S. parabrevigulonis n. sp. Isolate 1516 for the partial 18S (1707 bp, GenBank accession number KC875226), 28S D2-D3 (780 bp, KC875232), ITS1/2 (1050 bp, KC875227), of S. sucus isolate 10984 for the partial 18S (1708 bp, KC875229), 28S D2-D3 (780 bp, KC894755) and ITS1/2 (1059 bp, KC875230 and

1060 bp, KC875231) were determined. Alignment of the sequences with Clustal W resulted in datasets of 1791 characters for 18S, 807 characters for 28S D2-D3 and 1177 characters for ITS. Phylogenetic relationships among the isolates were determined separately for each dataset using Bayesian inference (BI) with Aphelenchus avenae as outgroup. The 50% majority rule consensus phylogenetic trees were generated from 18S and ITS1/2 dataset alignment by BI analysis under the GTR + I + G model, and from 28S D2-D3, under TrN + I + G model.

The trees based on sequences of 18S (Fig. 4-7) and 28S D2-D3 (Fig. 4-8) showed S. parabrevigulonis n. sp., S. sucus isolate 10984 and S. sucus isolate Chiyoda (Kanzaki & Tanaka, 2013) clustered into an independent clade among Bursaphelenchus species. Sheraphelenchus parabrevigulonis n. sp. shared a similar 18S sequence with S. sucus isolate Chiyoda (97.6% similarity) and for 28S D2-D3 similarity was 88.7%. Sheraphelenchus sucus isolate 10984 shared sequence similarity with S. sucus isolate Chiyoda for 18S (99.8%) and for 28S D2-D3 (99.8%). The low sequences divergence between these two S. sucus isolates revealed that intraspecific variation may exist and isolate 10984 is herein regarded as a geographical isolate and very similar to the type population of S. sucus isolate Chiyoda. The exceptionally large reported difference in stylet length is probably, as discussed above, the result of a fixation artefact in the original description.

The ITS1/2 tree (Fig.4-9) indicated S. parabrevigulonis n. sp. and S. sucus isolate 10984 clustered into an independent clade. Furthermore, the topology of three trees (Figs 4-7, 4-8, 4-9) also revealed that Sheraphelenchus species have a close relationship with Bursaphelenchus species, which may infer that these taxa could have shared a most recent common ancestor.

Fig. 4-7. Phylogenetic relationships of Sheraphelenchus and aphelenchid nematodes based on 18S. The 10 001st Bayesian tree inferred from 18S under GTR + I + G model (ln L = 15 710.5410; freqA = 0.2507; freqC = 0.1940; freqG = 0.2593; freqT = 0.2960; R(a) = 1.2691; R(b) = 2.8781; R(c) = 1.1276; R(d) = 0.8350; R(e) = 4.2440; R(f) = 1.0000; Pinvar = 0.2839; Shape = 0.5036). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Fig. 4-8. Phylogenetic relationships of Sheraphelenchus and aphelenchid nematodes based on partial 28S. The 10 001st Bayesian tree inferred from 28S D2-D3 under TrN + I + G model (ln L = 14 229.2148; freqA = 0.1949; freqC = 0.1680; freqG = 0.3072; freqT = 0.3299; R(a) = 1.0000; R(b) = 3.0230; R(c) = 1.0000; R(d) = 1.0000; R(e) = 4.2819; R(f) = 1.0000; Pinvar = 0.1360; Shape = 1.0480). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Fig. 4-9. Phylogenetic relationships of Sheraphelenchus and aphelenchid nematodes based on partial ITS1/2. The 10 001st Bayesian tree inferred from ITS1/2 under GTR + I + G model (ln L = 24 227.2969; freqA = 0.2634; freqC = 0.1887; freqG = 0.2329; freqT = 0.3150; R(a) = 1.1499; R(b) = 2.4916; R(c) = 1.3247; R(d) = 0.8831; R(e) = 3.0866; R(f) = 1.0000; Pinvar = 0.0814; Shape = 1.4797). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Discussion

Based on the morphology and morphometric data, S.parabrevigulonis n. sp. is most similar to S. brevigulonis (Massey & Hinds, 1970), the number of male subventral caudal papillae (three pairs vs two pairs) being the only clear reported difference between these two species. However, the hosts of these two species are completely different; S. parabrevigulonis n. sp. was isolated from pine wood packaging imported

75 from Italy, and S. brevigulonis was isolated from the necrotic tissues of Cenangium and Ceratocystis cankers of aspens in North America. As already mentioned, the original description of S. brevigulonis apparently lacks certain important information; the species has not been reported since its original description and molecular data are entirely lacking – hence the decision to name our isolate as a new taxon.

The redescribed isolate 10984 of S. sucus only differs from the type isolate Chiyoda by the reported stylet length of the male (13.5 (12.8-14.0) vs 6.5 (5.5-7.0) μm) and female (13.2 (12.3-13.9) vs 6.5 (5.5-7.0) μm), the possible reason for this being discussed above. According to the high sequences similarity of 99.8% for both 18S and 28S D2-D3 genes between these two S. sucus isolates, we conclude that they are conspecific. Sheraphelenchus species are commonly associated with nitidulid beetles, i.e., S. brevigulonis was isolated from the necrotic tissues of Cenangium and Ceratocystis cankers of aspens associated with Epurea sp. (Massey & Hinds, 1970), S. entomophagus was discovered from Carpophilus mutilates and Urophorus humeralis (Nickle, 1970), whilst S. sucus may be associated with nitidulid beetles found in association with the sap flow of Quercus trees (Kanzaki & Tanaka, 2013). In the present paper, S. parabrevigulonis n. sp. was isolated from pine wood packaging from Italy, which may have been incompletely treated according to ISPM15 (FAO, 2003). Before manufactured as packaging materials, the pine wood could have been infested by pine bark beetles, the eggs of which can be predated by a type of nitidulid beetle, Mimemodes japonus (Kishi, 1970). The redescribed isolate 10984 of S. sucus was obtained from onion bulbs from South Korea, which may have decomposed, thereby producing the volatiles that are known to attract nitidulid sap beetles (Nout & Bartelt, 1998). We may infer that both the S. parabrevigulonis n. sp. Wood packaging isolate and S. sucus onion isolate were possibly, maybe probably, associated with nitidulid beetles. Attempts to culture these two species on B. fuckeliana failed and so re-isolation of the nematodes and observations on their insect associations and host preferences are necessary to elucidate the life history of Sheraphelenchus species.

Kanzaki & Tanaka (2013) thought that Sheraphelenchus was probably, based upon molecular phylogeny, a junior synonym of Bursaphelenchus, although they refrained from formally making the proposal. However, our phylogenetic analyses revealed that S. parabrevigulonis n. sp. and S. sucus clustered into an independent clade and grouped with Bursaphelenchus species, which indicates that Sheraphelenchus is a

76 monophyletic group that may share a recent common ancestor with Bursaphelenchus. Among the species of Bursaphelenchus, only B. kiyoharai Kanzaki, Maehara, Aikawa, Masuya & Giblin-Davis, 2011 and B. posterovulvus Gu, Wang, He, Wang, Chen & Wang, 2014 showed a close similarity with Sheraphelenchus species in some similar morphological characters. For example, the male of B. kiyoharai possesses a similar genital papilla disposition and a spike-like tail as in Sheraphelenchus species, but the female, with V = 63.3-68.7, differs from that of Sheraphelenchus species where V is higher than 80. Bursaphelenchus posterovulvus is similar to Sheraphelenchus species only in the V value (82-86 vs >80). Furthermore, Hunt (1993) mentioned that Sheraphelenchus was characterised by the well-developed stylet lacking basal knobs or swellings. However, through the high resolution of photomicrographs, a stylet with slight basal swellings was clearly observed in S. parabrevigulonis n. sp. And both the S. sucus isolates 10984 and Chiyoda (Kanzaki & Tanaka, 2013). Sheraphelenchus parabrevigulonis n. sp. and the redescribed isolate 10984 of S. sucus have not only provided more detailed morphological and morphometric characters, but have expanded the molecular data available and confirmed the generic status of Sheraphelenchus.

Chapter V

Description of Pseudaphelenchus zhoushanensis n. sp. (Tylenchina: Aphelenchoididae) found in the wood of Pinus thunbergii at Zhoushan Islands, Zhejiang Province, China

Chapter published as:

Fang, Y. 1,2, Li, H.1, Maria, M. 1 and Bert, W2. (2014). Description of Pseudaphelenchus zhoushanensis n. sp. (Tylenchina: Aphelenchoididae) found in the wood of Pinus thunbergii at Zhoushan Islands, Zhejiang Province, China. Nematology 18, 1151-1164.

1Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R. China. 2Nematology Research Unit, Department of Biology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium.

Abstract

Pseudaphelenchus zhoushanensis n. sp. was isolated from a dead Pinus thunbergii at Changgang Mountain, Zhoushan Islands, Zhejiang Province, China. It is characterised by the small to medium length body, cuticle slightly annulated, presence of three lateral lines, stylet 9.0-10.7 μm with small but conspicuous basal knobs, excretory pore located from same level as the metacorpus to slightly anterior to metacorpus, true bursa surrounding entire tail but inconspicuous, male tail conical with a single mucron, spicule with distinct condylus and rostrum strongly arcuate to a pointed end, female tail conical with annulation, strongly ventrally bent in distal part of tail, with terminus bluntly pointed or finely mucronate. Phylogenetic analyses using sequences of the18S and 28S D2-D3 regions of rDNA confirmed the status of P. zhoushanensis n. sp. as a new species. Combining the molecular phylogenetic analyses, morphology and biology of P. zhoushanensis n. sp. and Tylaphelenchus jiaae indicates that T. jiaae is a member of Pseudaphelenchus to which it is herein transferred as P. jiaae n. comb. (= T. jiaae).

Keywords – molecular, morphology, morphometrics, new combination, new species, phylogeny, Pseudaphelenchus jiaae n. comb., SEM, taxonomy, Tylaphelenchus.

Introduction

Pseudaphelenchus Kanzaki & Giblin-Davis, 2009 in Kanzaki et al., 2009a currently comprises four species: P. yukiae Kanzaki & Giblin-Davis, 2009 in Kanzaki et al., 2009a, P. vindai Kanzaki & Giblin-Davis, 2010 in Kanzaki et al., 2010, P. scheffrahni Kanzaki, Li, Lan & GiblinDavis, 2014, and P. sui Kanzaki, Li, Lan & Giblin-Davis, 2014. Pseudaphelenchus was considered to be unique in having a tylenchid-like bursa and aphelenchoid-like pharynx (Kanzaki et al., 2014c). Initially Pseudaphelenchus was placed in the Aphelenchoidinae (Kanzaki et al., 2009a; 2010), but was later placed in Tylaphelenchinae based on the common typological characters of Pseudaphelenchus and Tylaphelenchus (Kanzaki et al., 2014c).

Previous to this paper, all Pseudaphelenchus species had been isolated from termites (Kanzaki et al., 2014c), but during our nematological survey of pine trees at Changgang Mountain, Zhoushan Islands, Zhejiang Province, China, an unknown species of Pseudaphelenchus was isolated from Pinus thunbergii Parl. 1868 wood samples. No termites were observed; only scolytid (bark beetles) tunnels occurred in the dead P. thunbergii tree. This is the first report of Pseudaphelenchus species isolated from P. thunbergii. Morphological and molecular characterisations revealed this species to be new and it is described herein as P. zhoushanensis n. sp.

Materials and methods

CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS

Sawn samples collected from a dead P. thunbergii tree were sliced into small chips ca 1 cm wide. The nematodes were isolated by the modified Baermann funnel technique for 24 h and multiplied successfully by picking five males and five females onto Botryotinia fuckeliana (de Bary) Whetz. growing on potato dextrose agar cultures for 4 weeks at 25°C. Adults for observation and measurements were isolated from fungal culture. Permanent slides were prepared using heat-killed nematodes fixed with FA 4:1 and ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). Morphometrics, drawings and light micrographs of nematodes were done with the aid of a Zeiss microscope equipped with a Zeiss AxioCam MRm CCD camera. For scanning electron microscopy (SEM), specimens were fixed with 2% paraformaldehyde (PFA) + 2.5% glutaraldehyde in 0.1 M Sorensen buffer, then

81 washed and dehydrated in ethanol solutions and subsequently critical point-dried with CO2. After mounting on stubs, the samples were coated with gold following the procedure detailed by Steel et al. (2011) and observed with a JSM-840 EM (JEOL) at 12 kV.

MOLECULAR ANALYSES

DNA samples were prepared according to Li et al. (2008). Three sets of primers (synthesised by Invitrogen) were used in the PCR analyses to amplify the near-full length 18S region and 28S D2-D3 expansion segments of ribosomal RNA genes (rDNA). The 18S region was amplified as two partially overlapping fragments; for the first fragment, 988F (5’-CTC AAA GAT TAA GCC ATG C-3’) and 1912R (5’-TTT ACG GTC AGA ACT AGG G-3’) were used and for the second fragment 1813F (5’- CTG CGT GAG AGG TGA AAT-3’) and 2646R (5’-GCT ACC TTG TTA CGA CTT TT-3’) (Holterman et al., 2006). The 28S D2-D3 region was amplified with the forward primer D2A (5’-ACA AGT ACC GTG AGG GAA AGT TG-3’) and the reverse primer D3B (5’- TCG GAA GGA ACC AGC TAC TA-3’) (De Ley et al., 1999). PCR conditions were as described by Ye et al. (2007) and Li et al. (2008). PCR products were separated on 1.5% agarose gels and visualised by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Invitrogen.

The near full length 18S and partial 28S D2-D3 sequences of rDNA gene of P. zhoushanensis n. sp. were compared with other aphelench species available in GenBank using the BLAST homology search program. The alignment of selected sequences was conducted by MAFFT (Katoh & Standley, 2013) with default parameters and edited by AliView (Larsson, 2014). The best fitted model of DNA evolution and the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were obtained using jModelTest2 (Darriba et al., 2012) with the Akaike Information Criterion (AIC). The tree topology for each gene was obtained separately using MrBayes 3.2.3 (Ronquist & Huelsenbeck, 2003) with four chains (three heated and one cold). The number of generations for the total analysis was set to 1 × 107, with the chain sampled every 1000 generations and the burn-in value was 25%. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using 50% majority rule (Larget & Simon, 1999). The consensus

82 trees were selected to represent the phylogenetic relationships with branch length and support level and visualised using TreeGraph 2 (Stöver & Müller, 2010).

Results

Pseudaphelenchus zhoushanensis* n. sp.

(Figs 5-1,5-2, 5-3, 5-4)

* Specific epithet derived from the type locality of the Zhoushan Islands.

MEASUREMENTS

See Table 5-1.

DESCRIPTION

Male

Body slender, J-shaped when heat-relaxed. Cuticle thin with weak annulation. Lateral field with three incisures. Head rounded, offset from body. Lip region flattened in lateral view, without annulation, about twice as broad as high. SEM en face observation showing a six-lobed head with distinct labial disc surrounded by a rim. Oral aperture rounded, diam. ca 0.2 μm, surrounded by six inner labial sensilla. Outer labial sensilla difficult to see. Amphidial apertures oval, positioned dorsolaterally on edges of labial plate of lateral lips, four cephalic papillae at same level as amphids on subdorsal and subventral lips. Stylet 9.0-10.7 μm with small but conspicuous basal knobs, conus forming ca 40% of stylet length. Procorpus cylindrical, ca 2.5 stylet lengths long. Metacorpus (media bulb) round, spherical, conspicuous valve plates situated centrally. Dorsal pharyngeal gland orifice opening into lumen of metacorpus ca one metacorpal valve length anterior to metacorpal valve. Pharyngo-intestinal junction immediately posterior to base of metacorpus. Nerve ring ca one stylet length posterior to metacorpus. Pharyngeal gland lobe ca 4-5 stylet lengths long, overlapping intestine dorsally. Excretory pore located at same level as middle of metacorpal valve plates to anterior to top of metacorpus. Hemizonid ca 4-5 stylet lengths posterior to base of metacorpus. Gonad on right side of intestine outstretched, reflexed in some individuals. Anterior part of testis containing developing spermatocytes in 3-5 rows, and then larger spermatocytes arranged in one to three rows in remainder of testis. Vas deferens sometimes containing developed sperm. Spicules paired, separate, condyles truncate sometimes broadly rounded, rostrum conical or triangular with bluntly

83 pointed tip, calomus smoothly tapering together with lamina, strongly arcuate in middle then sharply tapering towards pointed distal tip, cucullus absent. Gubernaculum absent. Tail conoid, ca 2.5-3.5 cloacal body diam. long, weakly arcuate with a short mucron (>90% of individuals). No single precloacal papilla (P1) observed. Three pairs of subventral papillae present: one pair subventral precloacal papillae (P2) located slightly anterior to cloacal slit, one pair subventral postcloacal papillae (P3) located at ca 75% of tail length posterior to cloacal slit, one tiny pair of papillae (P4) situated at base of tail terminus. Bursa narrow, long, not conspicuous, collapsed after fixation of SEM, starting ca one cloacal body diam. anterior to cloaca, surrounding entire tail.

Female

Body slender, slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region similar to male. Reproductive tract consisting of ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac. Ovary single, outstretched, developing oocytes arranged in multiple rows, several well developed oocytes arranged in 1-2 rows, a large elongated oocyte observed close to oviduct in some individuals. Oviduct short, tube-like. Spermatheca ca one vulval body diam. long, formed by small rounded cells, containing sperm in some individuals. Crustaformeria constructed of relatively large rounded cells. Uterus roundish, with thick wall. Vagina slightly inclined anteriorly. Vulva a simple slit in ventral view, without vulval membranes in lateral view, anterior and posterior lips slightly protruding. Postuterine sac ca 4-5 vulval body diam. long, extending for ca 50-65% of vulval-anus distance, sometimes filled with large sperm. Anus distinct, a dome-shaped slit in ventral view. Tail conical, 3.4-5.1 anal body diam. long, smoothly tapering and strongly ventrally bent in distal part of tail. Tail terminus with clear annulations and tip variable, e.g., fine or bluntly pointed, with or without mucro.

Table 5-1. Morphometrics of Pseudaphelenchus zhoushanensis n. sp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n - 20 20 L 401 427 ± 36.6 (344-499) 493 ± 41.7 (409-556) a 34.1 33.4 ± 2.5 (29.0-37.0) 31.3 ± 2.8 (25.3-36.4) b 8.7 9.0 ± 0.5 (7.8-9.5) 10.5 ± 0.6 (9.5-11.7) b' 4.1 4.6 ± 0.4 (4.1-5.4) 5.4 ± 0.5 (4.7-6.9) c 13.8 13.0 ± 0.7 (12.0-14.5) 13.7 ± 1.3 (11.4-16.7) c' 2.7 3.2 ± 0.3 (2.6-3.8) 4.3 ± 0.5 (3.4-5.1) V or T 46.2 52.4 ± 7.7 (40.1-67.6) 74.3 ± 1.6 (71.9-77.1) Body diam. 11.8 12.8 ± 0.7 (11.2-14.0) 16.0 ± 1.9 (13.5-20.4) Stylet length 9.3 9.7 ± 0.5 (9.0-10.7) 10.2 ± 0.5 (9.3-11.2) Lip diam. 4.7 5.0 ± 0.3 (4.6-5.6) 5.6 ± 0.3 (5.2-6.0) Lip height 2.4 2.5 ± 0.2 (2.1-2.7) 2.5 ± 0.2 (2.0-2.9) Lip diam./height 2.0 2.0 ± 0.1 (1.7-2.4) 2.2 ± 0.2 (1.9-2.6) Median bulb length 11.0 10.5 ± 0.7 (9.1-12.3) 11.5 ± 0.6 (9.8-12.4) Median bulb diam. 7.4 7.6 ± 0.4 (6.6-8.5) 9.1 ± 0.7 (7.7-10.9) Median bulb length/diam. 1.5 1.4 ± 0.1 (1.2-1.6) 1.3 ± 0.1 (1.1-1.4) Anterior end to excretory 36.2 39.7 ± 4.7 (28.1-47.5) 39.4 ± 3.8 (32.5-45.3) pore Anterior end to hemizonid 86.0 86.1 ± 8.6 (71.5-101.6) 83.7 ± 6.0 (72.6-96.6) Spicule (chord) 12.9 12.9 ± 0.9 (11.4-15.2) - Spicule (curved median line) 13.0 12.8 ± 0.9 (10.3-14.0) - Spicule (dorsal limb) 15.8 16.3 ± 1.1 (14.0-18.0) - Spicule (ventral limb) 11.2 11.5 ± 0.8 (9.1-12.9) - Ovary or testis length 185 224 ± 43.7 (152-293) 242 ± 61.8 (158-398) Post-uterine sac - - 55.6 ± 7.9 (39.8-66.3) Vulva to anus distance - - 93.0 ± 9.6 (74.3-115) Post-uterine sac length/ - - 60.9 ± 9.7 (41.1-79.7) vulva to anus (%) Tail length 29.0 32.9 ± 3.2 (27.2-39.3) 36.3 ± 3.9 (28.8-44.1) Cloacal or anal body diam. 10.7 10.4 ± 0.6 (8.5-11.3) 8.4 ± 0.8 (6.2-9.7)

Fig. 5-1. Pseudaphelenchus zhoushanensis n. sp. A: Entire female; B: Entire male; C: Stylet; D: Anterior part of female; E, F: Female reproductive tract, ovary (OV), oviduct (OD), spermatheca (SP), crustaformeria (CR), uterus (UT), vagina/vulva (V/V) and post-uterine sac are arranged from anterior; G: Lateral lines; H: Spicule; I: Lateral view of male tail; J: Ventral view of male tail; K, L: Female tail. Abbreviations: P + number = genital papillae. (Scale bars = 10 μm.)

Fig. 5-2. Light micrographs of Pseudaphelenchus zhoushanensis n. sp. male. A: Entire body; B: Anterior region showing excretory pore (arrow); C: Variation of position of excretory pore (arrow); D: Hemizonid in different focal planes; E: Male tail in ventral view showing caudal papillae and bursa; F-J: Lateral view of male tail in different focal planes; K: Lateral view of male tail without bursa. Abbreviations: P + number = caudal papillae. (Scale bars = 10 μm.)

Fig. 5-3. Light micrographs of Pseudaphelenchus zhoushanensis n. sp. female. A: Entire body; B: Anterior region; C: Lateral field; D: Female reproductive tract; E: En face view of head; F: Vulval region in lateral view; G, H: Female tail. (Scale bars = 10 μm.)

Fig. 5-4. Scanning electronic microscopy of Pseudaphelenchus zhoushanensis n. sp. A: En face view of female head; B: Head region; C: Vulval region in ventral view; D: Vulval region in lateral view; E: Lateral field; F: Ventral view of female tail; G, H: Female tail; I: Male tail terminus; J: Ventral view of male tail; K: Lateral view of male tail; L: Male tail. Abbreviations: Am = amphid; Cp = cephalic papilla; P + number = genital papillae. (Scale bars = 10 μm.)

TYPE HABITAT AND LOCALITY

The type specimens were collected from a culture of Botryotinia fuckeliana. The culture was started from five males and five females extracted from a dead P.

89 thunbergii tree in a forest on Changgang Mountain, Zhoushan Islands, Zhejiang Province, P.R. China (GPS: 30°01’46”N 122°07’09”E).

TYPE MATERIAL

Holotype male, 15 male and 16 female paratypes (slide numbers N06-1 to N06-7) deposited in the nematode collection of Nanjing Agricultural University, Nanjing, P.R. China. Five paratype males and four paratype females (slide numbers UGMD104314 and UGMD104315) deposited in the Ghent University Museum, Zoology Collection, Ghent, Belgium.

DIAGNOSIS AND RELATIONSHIPS

Pseudaphelenchus zhoushanensis n. sp. is characterized by the male 427 (344-499) μm and female 493 (409-556) μm body length and by the presence of three equal incisures in the lateral field. The slender stylet has small and conspicuous basal knobs. The excretory pore is located between the centre of the metacorpus to anterior to the anteriormost margin of the metacorpus or 40 (28-47) μm from the head. The spicules have a conspicuous capitulum, rounded to truncate condylus, conical rostrum with bluntly pointed tip and calomus smoothly tapering together with lamina, strongly arcuate in middle part then sharply tapering towards pointed distal tip. A long and narrow bursa surrounds the entire tail but is inconspicuous. The conical male tail has a short mucron in more than 90% of individuals. The female has a long and conical tail tapering to a variably shaped terminus.

There are four known species of Pseudaphelenchus, i.e., P. scheffrahni, P. sui, P. vindai and P. yukiae. Pseudaphelenchus zhoushanensis n. sp. can be distinguished from P. scheffrahni by the position of excretory pore (at same level of metacorpal valve plates to slightly anterior to metacorpus vs at same level as median bulb, i.e., from near centre of metacorpus to metacorpus base), bursa shape (narrow and relatively inconspicuous vs narrow but clearly discerned), male tail tip (bearing a short mucron vs mucron absent) and female tail tip (clearly annulated and tapering to bluntly pointed tip which is occasionally mucronate vs tail without annulation and a bluntly pointed or conical with irregularly rounded tip). The new species differs from P. sui by excretory pore position (at same level of metacorpal valve plates to slightly anterior to metacorpus vs at same level as median bulb, i.e., from near centre of metacorpus to metacorpus base), and the female tail tip (bluntly pointed, occasionally

90 bearing a short mucron vs bluntly or clearly pointed or conical tip). It differs from P. vindai by bursa shape (narrow and relatively inconspicuous vs clearly discerned) and male tail tip (bearing a short mucron with unclear bursa around the tip vs bluntly pointed with clear bursa surrounding the tip). It also differs from P. yukiae by the number of lateral lines (three vs four), the excretory pore position (at the same level of metacorpal valve plates to slightly anterior to metacorpus vs near metacorpus base), bursa shape (narrow and relatively inconspicuous vs large and clearly discerned) and spicule shape (well defined, arcuate, with distinct condylus and rostrum vs condylus and rostrum relatively undeveloped).

MOLECULAR PROFILES AND PHYLOGENETIC STATUS

Near-full-length sequences of the 18S region and D2-D3 28S expansion segments of rDNA gene were obtained with accession numbers KX155843 (1703 bp) and KX168423 (713 bp), respectively. The sequences were aligned by MAFFT and modified manually in datasets of 1912 characters for 18S and 1015 characters for 28S D2-D3. Phylogenetic relationships among the isolates for each dataset were determined using Bayesian inference (BI), with Aphelenchus avenae and Paraphelenchus acontioides as outgroups. The 50% majority rule consensus phylogenetic trees were generated from 18S and 28S D2-D3 dataset alignment to BI analysis both under the GTR + I + G model.

Based on the 18S and 28S D2-D3 gene sequences, P. zhoushanensis n. sp. clustered within the two morphologically most similar species: P. scheffrahni and P. sui, with high posterior probability support. The comparison of pairwise sequence revealed that the sequences of 18S and 28S D2-D3 domains for the new species with other similar species were largely different and the sequence divergences are marked in the tree (Fig. 5-5). The branch length of the Pseudaphelenchus clade was fairly long compared with other clades of genera in the Aphelenchoididae (see Supplementary Figs 5-S1, 5-S2).

The new species forms a well-supported molecular clade with Pseudaphelenchus spp. (P. scheffrahni, P. sui, P. vindai and P. yukiae) and T. jiaae which is in accordance with Kanzaki et al. (2014). However, according to the 18S tree topology, this basal clade is sister to all other Aphelenchoididae as in Kanzaki et al. (2014), whereas in the D2-D3 28S gene sequence tree, these two genera clade together with Aphelenchoides

91 huntensis Esmaeili, Fang, Li & Heydari, 2016, sister to an assemblage of five paraphyletic genera, Aphelenchoides Fischer, 1894, Laimaphelenchus Fuchs, 1937, Ficophagus Davies & Bartholomaeus, 2015 in Davies et al., 2015, Martininema Davies & Bartholomaeus, 2015 in Davies et al., 2015 and Schistonchus Cobb, 1927.

Pseudaphelenchus zhoushanensis n. sp. is sister to P. scheffrahni and P. sui in both the 18S and D2-D3 phylogenies, but the new species differs from these species by 178 bp and 192 bp, respectively, for the near-full length 18S, and 118 bp and 134 bp, respectively, for the 28S D2-D3 region nucleotides, thereby supporting its status as a new species.

Discussion

The diagnostic characters for Pseudaphelenchus were presented by Kanzaki et al. (2014) as: i) small to mediumsized species (<700 μm); ii) short (ca 10 μm) stylet with small but clear basal knobs; iii) spherical median bulb; iv) separate male spicules of various shapes; v) male tail conoid with long bursa supported by genital papillae; vi) female tail conoid with variable terminus; and vii) associated with termites. However, this new species might be linked with scolytid beetles of pine trees rather than termites. Being associated with termites and the presence of a bursa (vs association with wood-boring beetles, scolytids, weevils and cerambycids and absence of bursa) have been cited as being the main differences between Pseudaphelenchus and Tylaphelenchus Rühm, 1956 (Kanzaki et al., 2014c). However, phylogenetic analyses suggested that T. jiaae is embedded within the currently described and sequenced Pseudaphelenchus species and SEM comparison between P. zhoushanensis n. sp. and T. jiaae revealed both species are remarkably similar; a similar head region, similar genital papillae shape and arrangement, and similar bursa.

According to the diagnostic characters of Tylaphelenchus presented by Hunt (1993) and Kanzaki et al. (2014), the stylet is short and robust with large and rounded knobs, and the male tail bears 1-3 mucrons and is without a bursa. Indeed, all the valid species of Tylaphelenchus, T. leichenicola Rühm, 1956 (type species), T. christinae Lieutier & Laumond, 1978, T. georgiensis Devdariani, 1970, T. grossmannae Rühm, 1965 and T. paramonovi Kakulia, 1963, have a robust stylet with typically large rounded knobs.

Fig. 5-5. Phylogenetic relationships of Pseudaphelenchus zhoushanensis n. sp. and aphelenchid nematodes based on combining near-full-length of 18S and 28S D2-D3 region. Several sequences of genera or same species were shrank to a triangle clade. The pairwise sequences divergence of the new species and closely related species are given.

Fig. 5-S1. Phylogenetic relationships of Pseudaphelenchus zhoushanensis n. sp. and aphelenchid nematodes based on near full length of 18S. The 10 001st Bayesian tree inferred from 18S under GTR + I + G model (ln L = -37 515.4876; freqA = 0.2430; freqC = 0.1869; freqG = 0.2654; freqT = 0.3047; R(a) = 1.2824; R(b) = 2.8772; R(c) = 1.2818; R(d) = 1.0870; R(e) = 4.1881; R(f) = 1.0000; Pinvar = 0.1530; Shape = 0.5560). Aphelenchus avenae and Paraphelenchus acontioides served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Fig. 5-S2. Phylogenetic relationships of Pseudaphelenchus zhoushanensis n. sp. and aphelenchid nematodes based on partial 28S D2-D3 region. The 10 001st Bayesian tree inferred from 28S D2-D3 region under GTR + I + G model (ln L = -42 842.1341; freqA = 0.1860; freqC = 0.1605; freqG = 0.3237; freqT = 0.3298; R(a) = 0.9431; R(b) = 3.7975; R(c) = 1.5186; R(d) = 0.7892; R(e) = 5.3502; R(f) = 1.0000; Pinvar = 0.1380; Shape = 0.6980). Aphelenchus avenae and Paraphelenchus acontioides served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

However, the stylet of T. jiaae is thin with small basal knobs or bipartite swellings and the male tail is without a mucron. Furthermore, albeit being described as absent in the original description (Huang et al., 2012), careful observation of SEM and LM images reveals the presence of an inconspicuous bursa covering the tail (original description Fig. 2F, G and Fig. 3H, I). Also, in P. zhoushanensis n. sp., the bursa is only vaguely seen and is collapsed after SEM fixation. Based on morphological and phylogenetic characterisation, we propose to transfer T. jiaae to the genus Pseudaphelenchus as P. jiaae (Huang, Ye, Liang, Lu & Zhang, 2012) comb. n. According to this update, the comparative diagnostic characters of genera Pseudaphelenchus and Tylaphelenchus are listed in Table 2.

Table 5-2. Comparative diagnostic characters of genus Pseudaphelenchus and Tylaphelenchus.

Characters Tylaphelenchus Pseudaphelenchus Stylet Short(ca 10 µm ) and robust with large Short (ca 10 µm ) and slender and rounded knobs stylet with small but clear basal knobs Spicules Separate and strongly curved with a Various shapes small condylus and rostrum Male tail Conoid with 1-3 mucron(s) Conoid with 1 mucron Bursa Absent Long and covering the whole tail, narrow or inconspicuous in some species Female tail Conoid with 1-3 mucron(s) Conoid with variable terminus Host Wood-boring beetles (scolytids, Termites or wood-boring beetles weevils and cerambycids) (scolytids)

At present, little is known about life cycles and biological association of Pseudaphelenchus. It has been assumed that insect association is probably one of general phoresy where the nematodes are transmitted to newly established termite colonies by vectors because individual nematodes have been found in termites (Kanzaki et al., 2014c) or in possible association with scolytids on dead pine trees (this study). However, the exact life cycle is not yet known and more detailed studies are required to shed light on the relationship and ecology of these species.

Chapter VI

Description of five unknown Aphelenchoides species

Manuscript in preparation:

Fang, Y. 1,2,3, Li, H.1*, Couvreur, M2. and Bert, W2.

3Technical Centre, Ningbo Entry-Exit Inspection and Quarantine Bureau, 9 Mayuan Road, Ningbo 315012, Zhejiang, P.R. China.

Abstract

Six Aphelenchoides populations, namely Aphelenchoides sp. 12Asp, Aphelenchoides sp. 14Asp, Aphelenchoides sp. 18Asp, Aphelenchoides sp. AspX and Aphelenchoides sp. Asp25 were isolated from different habitats from different countries and identified as five different unknown species based on morphological and molecular characters. All these unknown species belong to the Group 2 according to the category of Aphelenchoides species with the female tail shapes having a single mucro. The male of Aphelenchoides sp. 18Asp is characterised by spicules with a bifurcated distal tip, a unique trait for Aphelenchoides species. The large number of species in Group 2 makes the diagnosis of these five unknown speices difficult. The phylogenetic analysis of Aphelenchoides species based on rDNA 18S and 28S D2-D3 sequences confirmed the status of these five species as unkown species.

Keywords – molecular, morphology, morphometrics, phylogeny, Aphelenchoides, SEM, taxonomy.

Introduction

Aphelenchoides Fischer 1894 is a genus with over 200 species (Sánchez-Monge, 2016). Shahina (1996) divided Aphelenchoides species into four groups by the shape of tail terminus: Group 1, tail simple without any outgrowth or mucronated structure; Group 2, tail with one mucronated structure; Group 3, tail with tetramucaronate spine or star shape; and Group 4, tail outgrowth other than spine star. A comparison of these four groups reveals that the members of group 2 show the highest variation.

Between 2008 and 2017, numerous Aphelenchoides populations were detected in different habits, for example pine wood packaging materials and soil from different plants. Except for some populations with clear diagnostic characters and molecular data, i.e., A. unisexus, A. bicaudatus, A. subtenuis and A. besseyi, most of these populations belonged to group 2 sensu Shahina (1996). Due to the high morphological variability and overlap of characteristics in Aphelenchoides and several species that were not described in detail in the past and evidently without molecular data , it is especially difficult to recognize the many species based on old descriptions (Hockland 2001).

In order to identify and describe Aphelenchoides species accurately, Sánchez-Monge (2016) encouraged new taxa description with: i) Measurements of at least 15 females (and 5 males if possible); ii) Detailed drawings and detailed illustrations of particular features, especially the anterior and tail regions; iii) SEM images; iv) Molecular data of at least two specimens, preferably from two molecular markers and v) Virtual morphological vouchers deposited in a public repository. In this study, we followed these sugestions to diagnose six Aphelenchoides populations, which belong to group 2, and characterize them as five new species.

Materials and methods

CULTURING OF NEMATODE AND MORPHOLOGICAL OBSERVATIONS

The nematodes were isolated by the modified Baermann funnel technique for 24 h and multiplied successfully by picking five males and five females onto Botryotinia fuckeliana (de Bary) Whetz., growing on potato dextrose agar cultures for 4 weeks at 25°C. Adults for observation and measurements were isolated from this fungal culture. Permanent slides were prepared using heat-killed nematodes fixed with FA 4:1 and

99 ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). Morphometrics, drawings and light micrographs of nematodes were done with the aid of a Olympus BX51 DIC Microscope (Olympus Optical), equipped with an Olympus DP72camera for photography. For scanning electron microscopy (SEM), specimens were fixed with 2% paraformaldehyde (PFA) + 2.5% glutaraldehyde in 0.1 M Sorensen buffer. A subsequent ultrasonic treatment (8 min) of the specimens in a single drop of water removed particles adhering to the body surface. Then the specimens were dehydrated by passing them through a graded ethanol concentration series of 20, 50, 75, 95, 100% (1 h each), 100% (overnight) and 100% (20 min, next morning). Afterwards, the specimens were critical point-dried with liquid CO2, mounted on stubs with carbon discs and coated with gold (25 nm) before observation with a JSM-840 EM (JEOL, Tokyo, Japan) at 12 kV.

MOLECULAR ANALYSES

DNA samples were prepared according to Li et al. (2008). Three sets of primers (synthesised by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify sequences of the near full length 18S region and 28S D2-D3 expansion segments of ribosomal RNA genes (rDNA). The 18S region was amplified as two partially overlapping fragments, for the first fragment, the 988F (5’-CTC AAA GAT TAA GCC ATG C-3’) and 1912R (5’-TTT ACG GTC AGA ACT AGG G-3’), while for the second fragment, 1813F (5’-CTG CGT GAG AGG TGA AAT-3’) and 2646R (5’-GCT ACC TTG TTA CGA CTT TT-3’) were used (Holterman et al., 2006). The 28S D2-D3 region was amplified with the forward primer D2A (5’- ACA AGT ACC GTG AGG GAA AGT TG-3’) and the reverse primer D3B (5’-TCG GAA GGA ACC AGC TAC TA-3’) (De Ley et al., 1999). PCR conditions were as described by Li et al. (2008) and Ye et al. (2007). PCR products were separated on 1.5% agarose gels and visualised by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Invitrogen, Shanghai, China. The near full length 18S and partial 28S D2-D3 sequences of rDNA gene of Aphelenchoides species compared with other aphelenchids species available in GenBank using BLAST homology search program. The alignment of selected sequences were conducted by MAFFT (Katoh & Standley, 2013) with default parameters and edited by AliView (Larsson, 2014). The best fitted model of DNA evolution and the base frequency, the proportion of invariable sites, and the gamma

100 distribution shape parameters and substitution rates were obtained using jModelTest2 (Darriba et al., 2012) with the Akaike Information Criterion (AIC). The tree topology for each gene was obtained separately using MrBayes 3.2.3 (Ronquist & Huelsenbeck, 2003) with four chains (three heated and one cold). The number of generations for the total analysis was set to 10 million, with the chain sampled every 1000 generations and the burn-in value was 25%. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using 50% majority rule (Larget & Simon, 1999). The consensus trees were selected to represent the phylogenetic relationships with branch length and support level and visualised using TreeGraph 2 (Stöver & Müller, 2010).

Results

Six Aphelenchoides populations were identified as 5 species (table 1).

Table 6-1. Information of six Aphelenchoides populations.

Species identification Host Origin Reference Code Aphelenchoides sp. 12Asp Pinus thunbergii Shandong, China AspSDCD Acer palmatum Japan Asp6266 Aphelenchoides sp. 14Asp Pinus spp. Hongkong AspHK Aphelenchoides sp. 18Asp Oryza sativa Jiangsu, China AspJSYZ Aphelenchoides sp. AspX Pinus massoniana Jiangsu, China AspJSYX Lycopersicon Aphelenchoides sp. Asp25 Shandong, China AspSDSG esculentum

101

Aphelenchoides sp. 12Asp

(Figs 6-1, 6-2)

MEASUREMENTS

See Table 6-2.

DESCRIPTION

Male

Body slender, cylindrical, J-shaped when heat-relaxed. Cuticle with fine annulations. Lateral field with four incisures (i.e., three ridges with equal width), occasionally six only seen under SEM. Lip region rounded in lateral view, slightly offset, with clear annulation, about twice as broad as high. SEM en face observation showing a six-lobed head with six equally sized and partial fused cephalic sectors. Labial disc present with a circular groove. Oral aperture rounded, diam. ca 0.2 μm, surrounded by six slightly elevated inner labial sensilla. Outer labial sensilla hexagram-shaped. Amphidial apertures oval, positioned dorsolaterally on edges of labial plate of lateral lips, four cephalic papillae at same level as amphidial apertures on subdorsal and subventral lips. Stylet 9.6-11.6 μm long, with conspicuous basal swellings, conus occupying ca 40% of its total length. Procorpus cylindrical, ca 2.5 body diam. long. Metacorpus (median bulb) strongly developed, almost spherical to oval, with centrally situated valves. Dorsal pharyngeal gland orifice opening into lumen of metacorpus, ca one metacorpal valve length anterior to metacorpal valve. Pharyngo-intestinal junction immediately posterior to base of metacorpus. Nerve ring ca half body diam. length posterior to metacorpus. Pharyngeal gland lobe slender, ca 2.5-3 body diam. long, overlapping intestine dorsally. Excretory pore located at same level with nerve ring or anterior to nerve ring, but posterior to the posteriormost of metacorpus. Hemizonid ca 1 body diam. posterior to excretory pore, but occasionally immediately posterior to excretory pore. Testis single, located on left side of intestine, outstretched or anteriorly reflexed in some individuals, developing spermatocytes in one single column. Anterior part of testis containing developing spermatocytes in single or double rows, and then larger spermatocytes arranged in one single column. Posterior part of testis ca 3-4 body diam. long, forming vas deferens, containing several well developed sperm. Posterior end of vas deferens is fused with rectum to form a narrow

102 cloacal tube. Lips of cloacal opening slightly protruding. Spicules, separate, smoothly arcuate, rose-thorn shaped with rounded apex and rostrum, dorsal limb and ventral limb smoothly curved towards bluntly pointed distal tip, cucullus absent. Gubernaculum absent. Tail conoid, ca 3.0-4.0 cloacal body diam. long, possessing pointed tip bearing a short mucro ca 1.0–2.0 μm long and with tiny nodular protuberances. Three pairs of subventral papillae present: one pair subventral precloacal papillae (P2) located slightly anterior to cloacal slit, one pair subventral postcloacal papillae (P3) located at ca 60% of tail length posterior to cloacal slit, one tiny pair of papillae (P4) situated at base of tail terminus. Bursa absent.

Female

Body slender, cylindrical, slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region similar to male. Reproductive system single, ventrally located. Organs arranged as ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac. Ovary single, outstretched, anteriorly with double flexure in some individuals, developing oocytes arranged in single row, several well developed oocytes arranged in single row, a large elongated oocyte observed close to oviduct in some individuals. Oviduct short and connected with spermatheca, a rounded to oval sac ca 1.5-2.5 body diam. long filled with rounded sperm cells. Crustaformeria constructed of relatively large rounded cells. Uterus thick-walled, sometimes containing one developed egg. Vagina slightly inclined anteriorly to body axis. Vulva a simple slit in ventral view, without vulval membranes in ventral and lateral view, anterior and posterior lips slightly protruding. Post-uterine sac ca 2.5-3.5 vulval body diam. long, extending for ca 35-55% of vulval-anus distance, sometimes filled with large sperm. Anus distinct, a dome-shaped slit in ventral view. Tail conoid, ca 3-3.5 anal body diam. long. with hemispherical tail terminus bearing single mucron ca 1.0-2.0 µm long with tiny nodular protuberances in central

Table 6-2. Morphometrics of Aphelenchoides sp. 12Asp isolated from Pinus thunbergii in Changdao, Shandong Province and Acer palmatum imported from Japan. All measurements are in μm and in the form: mean ± s.d. (range).

Character AspSDCD Asp6266 Male Female Male Female Holotype Paratypes Paratypes n - 20 20 15 15

103

507 ± 16.1 636 ± 49.6 612 ± 27.9 713 ± 36.7 L 513 (471-537) (491-715) (567-668) (644-766) 29.4 ± 1.0 30.5 ± 1.2 37.1 ± 2.6 37.2 ± 2.0 a 29.6 (27.4-31.9) (28.1-32.8) (33.5-43.5) (33.7-41.0) 8.7 ± 0.38 10.2 ± 0.6 8.8 ± 0.5 10.1 ± 0.5 b 8.9 (8.11-9.79) (8.9-11.3) (7.9-9.6) (9.1-10.9) 4.5 ± 0.15 5.4 ± 0.3 5.1 ± 0.3 5.7 ± 0.3 b' 4.8 (4.2-4.8) (4.8-6.0) (4.7-5.9) (5.2-6.3) 17.3 ± 0.5 15.7 ± 1.0 20.0 ± 1.7 16.9 ± 0.9 c 17.7 (16.2-18.5) (13.9-18.1) (18.0-24.3) (14.9-18.2) 1.7 ± 0.1 3.5 ± 0.2 2.5 ± 0.2 3.8 ± 0.3 c' 1.6 (1.5-1.8) (3.2-4.1) (2.2-2.8) (3.3-4.3) 56.8 ± 3.9 66.7 ± 0.7 47.9 ± 3.5 66.8 ± 1.0 T or V 58.3 (44.5-63.9) (65.0-68.2) (43.4-58.3) (64.0-67.9) 17.3 ± 0.7 20.8 ± 1.6 16.6 ± 1.2 19.0 ± 1.8 Body diam. 17.3 (15.7-18.5) (16.7-22.8) (14.1-17.8) (16.0-22.7) 10.2 ± 0.4 10.5 ± 0.5 10.3 ± 0.4 10.6 ± 0.4 Stylet length 10.1 (9.6-11.6) (9.7-11.5) (9.9-11.2) (10.1-11.5) 5.5 ± 0.2 6.0 ± 0.3 Lip diam. 5.6 - - (5.0-6.0) (5.6-6.7) 2.5 ± 0.1 2.7 ± 0.1 Lip height 2.6 - - (2.3-2.7) (2.4-3.0) 2.2 ± 0.1 2.3 ± 0.1 Lip diam./height 2.2 - - (2.0-2.5) (2.0-2.5) 11.3 ± 0.5 12.5 ± 0.8 Median bulb length 11.3 - - (9.9-12.0) (10.5-14.5) 9.2 ± 0.4 10.7 ± 0.7 Median bulb diam. 10.0 - - (8.3-10.0) (8.9-11.9) Median bulb 1.2 ± 0.1 1.2 ± 0.0 1.1 - - length/diam. (1.1-1.4) (1.1-1.3) Anterior end to 63.7 ± 3.2 69.2 ± 5.2 73.0 ± 3.1 76.6 ± 3.4 60.6 excretory pore (57.6-70.6) (60.1-79.4) (67.2-78.3) (71.7-83.0) Anterior end to 82.9 ± 6.8 87.3 ± 4.9 73.1 - - hemizonid (71.9-94.6) (80.2-94.9) 12.8 ± 0.6 - 12.8 ± 1.0 Spicule (chord) 11.8 - (11.2-13.6) (10.4-14.2) Spicule (curved 11.3 ± 0.7 10.5 - - - median line) (10.0-12.3) 16.4 ± 0.8 Spicule (dorsal limb) 15.4 - - - (15.1-17.7) 8.0 ± 0.7 Spicule (ventral limb) 8.7 - - - (7.0-9.6) 287 ± 21.6 291 ± 53.0 293 ± 34.5 289 ± 49.0 Ovary or testis length 299 (222-317) (205-388) (237-381) (200-352) 69.6 ± 11.8 69.0 ± 7.1 Post-uterine sac - - - (51.3-95.8) (54.5-77.3) 18.4 ± 1.4 Vulva diam - - - - (14.6-20.7) Vulva to anus 170.1 ±14.9 - - - - distance (129-199) Post-uterine sac 40.9 ± 5.6 36.2 ± 2.6 length/Vulva to - - - (32.2-55.6) (30.5-40.4) anus(%) 29.3 ± 0.7 40.2 ± 2.7 30.7 ± 2.4 42.4± 1.2 Tail length 29.0 (27.6-30.8) (32.6-44.7) (25.2-33.7) (40.5-44.4) Cloacal or anal body 11.4 ± 0.6 11.4 ± 0.9 12.2 ± 0.8 11.2 ± 0.9 11.7 diam. (10.1-12.3) (9.3-12.6) (10.9-13.7) (9.7-12.8)

104

Fig. 6-1. Light photomicrographs of Aphelenchoides sp. 12Asp A: Entire female and male; B: Head region; C: Lateral lines; D: Vulval region; E-G: Male tails; H-J: Female tails. (Scale bars = 20 μm)

105

Fig. 6-2. Scanning electronic microscopy of Aphelenchoides sp. 12Asp A: En face view of female head; B: Head region of female; C: Lateral view of female tail; D: Ventral view of female tail; E: Vulval region in ventral view; F: Vulval region in lateral view; G: Female tail; H: Head region of male; I: Variation of Lateral field; J: Lateral view of male tail; K: Ventral view of male tail.

106

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides sp. 12Asp is characterized by body lengths of 507 (471-537) μm and 636 (491-715) μm for the male and female, respectively. The cuticle is weakly annulated and there are four (occasionally six) incisures with equal width in the lateral field. The labial disc is clearly defined by a circular groove with hexagram shape outer labial sensillae. The stylet is 10 (10-12) μm with small basal swellings. The excretory pore is located between the nerve ring to the posteriomost margin of the metacorpus or 64 (57-71) μm from the head. The spicules are smooth, curved, rose-thorn shaped with rounded apex and rostrum, dorsal limb smooth curved towards a hook distal tip. The male tail bears six (2 + 2 + 2) caudal papillae ending in a pointed mucron with tiny nodular protuberances. The spermatheca is rounded to oval and contains rounded sperm. The female tail is conoid with hemispherical tail terminus bearing a mucron with tiny nodular protuberances situated centrally. According to the categorization codes of OEPP/EPPO (2004), Aphelenchoides sp. 12Asp is defined as A2-B2-C1-D1-E1-F1/2.

The specimens of Aphelenchoides sp. 12Asp were isolated from a dead Pinus thunbergii in China. The Aphelenchoides sp. Asp6266 were isolated from a soil sample of Acer palmatum exported from Japan. Morphological and morphometric data of Aphelenchoides sp. 12Asp, Aphelenchoides sp. Asp6266 was very similar to Aphelenchoides sp. 12Asp, the major apparent differences being the female ratio a of 37.2 (33.7-41.0) vs 30.5 (28.1-32.8) and male ratio a of 37.1 (33.5-43.5) vs 29.4 (27.4-31.9).

Aphelenchoides sp. 12Asp has a female tail terminus with tiny nodular protuberances. According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 2, which is defined as having the female tail terminus with “one or sometimes two mucronate structures”. Based on the number of lateral lines, the stylet length and the female conoid or dorsally convex-conoid tail, it is close to fifteen species from Group 2, including A. absari Husain & Khan, 1967, A. brassicae Edward & Misra, 1969, A. daubichaensis Eroshenko, 1968a, A. echinocaudatus Haque, 1968, A. ensete Swart, Bogale & Tiedt, 2000, A. graminis Baranovskaya & Haque, 1968, A. haguei Maslen, 1979, A. macronucleatus Baranovskaya, 1963, A. nechaleos Hooper & Ibrahim, 1994, A. paranechaleos Hooper & Ibrahim, 1994, A. platycephalus Eroshenko, 1968b, A. richardsoni Grewal, Siddiqi & Atkey, 1992, A.

107 tsalolikhini Ryss, 1993, A. tuzeti B’Chir, 1979, and A. xui Wang, Wang, Gu, Wang, and Li, 2013.

The new species differs from A. absari by the longer female body length = 636 (491-715) vs (390-450) μm, longer male body length = 507 (471-537) vs (320-430) μm, higher female ratio b = 10.2 (8.9-11.3) vs (4.0-4.5), higher male ratio b = 8.7 (8.1-9.8) vs (4.3-5.2) and longer female tail length = 40.2 (32.6-44.7) vs 25 μm. It differs from A. brassicae by the higher female ratio c’ = 3.5 (3.2-4.1) vs 2.75, longer female tail length = 40.2 (32.6-44.7) vs 21.5 μm. It differs from A. daubichaensis by the presences of male. It differs from A. echinocaudatus by the longer female body length = 636 (491-715) vs 427 μm, higher female ratio a = 30.5 (28.1-32.8) vs 24.5, higher female ratio b = 10.2 (8.9-11.3) vs 8, higher female ratio c’ = 3.5 (3.2-4.1) vs 2.7 and longer female tail length = 40.2 (32.6-44.7) vs 22.8 μm. It differs from A. ensete by the labial disc (clear defined with a circular groove vs clearly defined without a circular groove), lower female ratio a = 30.5 (28.1-32.8) vs 48.4 (36.9-61.8) and lower male ratio a = 29.4 (27.4-31.9) vs 49.2 (41.7-58.1). It differs from A. graminis by longer female tail length = 40.2 (32.6-44.7) vs 24.4 μm. It differs from A. haguei by the shape of spicules (condyles round vs condyles with curved). It differs from A. macronucleatus the presences of male. It differs from A. nechaleos by the lower female ratio a = 30.5 (28.1-32.8) vs 41 (33-45), lower male ratio a = 29.4 (27.4-31.9) vs 41 (35-44) and shape of spicule (dorsal limb smoothly curved towards pointed distal tip vs dorsal limb convex curved towards a tiny hook distal tip). It differs from A. paranechaleos by the lower female ratio a = 30.5 (28.1-32.8) vs 43 (37-46), lower male ratio a = 29.4 (27.4-31.9) vs 45 (40-50), shape of spicule (dorsal limb smoothly curved towards pointed distal tip vs dorsal limb convex curved towards a tiny hook distal tip) and position of (P3) subventral postcloacal papillae (ca 60% of tail length posterior to cloacal slit vs at mid of tail length). It differs from A. platycephalus by the longer female body length = 636 (491-715) vs (245-268) μm, male body length = 507 (471-537) vs 245 μm, higher female ratio a = 30.5 (28.1-32.8) vs 24-27, higher male ratio a = 29.4 (27.4-31.9) vs 24.5, higher female ratio b = 10.2 (8.9-11.3) vs (5.1-6.2), higher male ratio b = 8.7 (8.1-9.8) vs 6.1, higher female ratio c = 15.7 (13.9-18.1) vs 9.1, higher male ratio c = 17.3 (16.2-18.5) vs 10 and position of excretory pore (at same level with nerve ring or anterior to nerve ring, but posterior to the posteriormost of metacorpus vs anterior to the anteriormost of metacorpus). It

108 differs from A. richardsoni by the presences of males. It differs from A. tsalolikhini by the higher female ratio b = 10.2 (8.9-11.3) vs 6.7 (6.4-7.4), higher male ratio b = 8.7 (8.1-9.8) vs 5.8 (5.3-6.5) and absences of two pairs of dark-stained sclerotized cells on the dorsal side of vagina. It differs from A. tumulicaudatus by the higher female ratio b = 10.2 (8.9-11.3) vs (3.9-5.5), higher male ratio b = 8.7 (8.1-9.8) vs (4.9-6.0) and lower male ratio a = 29.7 (27.3-32.7) vs 45 (40.7-42.9). It differs from A. tuzeti by the presence of males. It differs from A. xui by the head region (faint double groove on labial disc vs double groove on lip sectors) and shape of spicules (dorsal limb smoothly curved towards pointed distal tip vs dorsal limb convex curved towards a tiny hook distal tip).

Aphelenchoides sp. 14Asp

(Figs 6-3, 6-4)

MEASUREMENTS

See Table 6-3.

DESCRIPTION

Male

Body slender, cylindrical, J-shaped when heat-relaxed. Cuticle with fine annulations. Lateral field with four incisures (i.e., three ridges of equal width). Lip region rounded in lateral view, slightly offset, with clear annulation, about twice as broad as high. SEM en face observation showing a six-lobed head with six equally sized and partial fused cephalic sectors. Labial disc clear defined with a circular groove. Oral aperture rounded, diam. ca 0.2 μm, surrounded by six slightly elevated inner labial sensilla. Outer labial sensilla hexagram-shaped. Amphidial apertures oval, positioned dorsolaterally on edges of labial plate of lateral lips, four cephalic papillae at same level as amphidial apertures on subdorsal and subventral lips. Stylet 9.9-11.7 μm long, with conspicuous basal swellings, conus occupying ca 40% of its total length. Procorpus cylindrical, ca 2 body diam. long. Metacorpus (median bulb) strongly developed, almost spherical or oval, with centrally situated valves. Dorsal pharyngeal gland orifice opening into lumen of metacorpus ca one metacorpal valve length anterior to metacorpal valve. Pharyngo-intestinal junction immediately posterior to base of metacorpus. Nerve ring ca half body diam. length posterior to metacorpus. Pharyngeal gland lobe slender, ca 2-2.5 body diam. long, overlapping intestine

109 dorsally. Excretory pore located at same level with nerve ring or anterior to nerve ring to slightly anterior to posteriomost margin of the metacorpus. Hemizonid ca one body diam. posterior to excretory pore. Testis single, located on left side of intestine, outstretched or anteriorly reflexed in some individuals. Anterior part of testis containing developing spermatocytes in one single row, and then larger spermatocytes arranged in one single column. Posterior part of testis ca 4-5 body diam. long, forming vas deferens, containing several well developed sperm. Posterior end of vas deferens is fused with rectum to form a narrow cloacal tube. Lips of cloacal opening slightly protruding. Spicules separate, rose-thorn shaped with rounded apex and rostrum, dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip, ventral limb convex curved and ending slightly before the dorsal limb, cucullus absent. Gubernaculum absent. Tail conoid, ca 3.5-4.5 cloacal body diam. long, possessing pointed tip bearing a pointed mucro ca 2.0–2.5 μm long without tiny nodular protuberances. Three pairs of subventral papillae present: one pair subventral precloacal papillae (P2) located slightly anterior to cloacal slit, one pair subventral postcloacal papillae (P3) located at mid of the tail, one tiny pair of papillae (P4) situated at base of tail terminus. Bursa absent.

Female

Body slender, cylindrical, slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region similar to male. Reproductive system single, ventrally located. Including ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac. Ovary single, outstretched, anteriorly with double flexure in some individuals, developing oocytes arranged in single row, several well developed oocytes arranged in single row, a large elongated oocyte observed close to oviduct in some individuals. Oviduct short and connected with spermatheca, an axial and oblong sac ca 2-3 body diam. long filled with disc-like sperm cells. Crustaformeria constructed of relatively large rounded cells. Uterus thick-walled, sometimes containing one developed egg. Vagina slightly inclined anteriorly to body axis. Vulva a simple slit in ventral view, without vulval membranes in ventral and lateral view, anterior and posterior lips slightly protruding. Post-uterine sac ca 2-3 vulval body diam. long, extending for ca 40-50% of vulval-anus distance, sometimes filled with large sperm. Anus distinct, a dome-shaped slit in ventral view. Tail conoid or dorsally convex-conoid, ca 3.5-4.5 anal body diam. long. with step-like projection ca 1.0-2.0

110

µm long with many tiny nodular protuberances in ventral.

Table 6-3. Morphometrics of Aphelenchoides sp. 14Asp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n - 20 20 L 650 634 ± 76.2 (447-760) 683 ± 66.0 (581-802) a 29.9 29.7 ± 1.6 (27.3-32.7) 28.8 ± 1.9 (25.3-32.7) b 8.6 9.7 ± 1.1 (7.1-11.9) 10.5 ± 0.9 (9.2-12.1) b' 4.9 5.0 ± 0.5 (3.8-5.9) 5.4 ± 0.5 (4.3-6.7) c 16.3 16.6 ± 1.5 (13.2-19.7) 15.5 ± 1.2 (13.4-17.8) c' 2.4 2.7 ± 0.3 (2.2-3.3) 3.9 ± 0.3 (3.4-4.5) V or T 69.1 68.7 ± 1.5 (63.6-70.7) 59.8 ± 9.7 (46.0-82.9) Body diam. 21.7 21.4 ± 2.5 (14.1-25.1) 23.7 ± 2.3 (19.2-28.7) Stylet length 11.7 11.1 ± 0.5 (10.0-11.7) 11.5 ± 0.5 (10.5-12.3) Lip diam. 6.9 6.6 ± 0.4 (5.5-7.1) 7.0 ± 0.3 (6.5-7.9) Lip height 2.6 2.8 ± 0.2 (2.4-3.3) 2.9 ± 0.2 (2.5-3.3) Lip diam./height 2.7 2.3 ± 0.2 (1.9-2.7) 2.4 ± 0.2 (2.1-2.8) Median bulb length 15.1 13.6 ± 1.0 (11.1-15.1) 15.1 ± 1.1 (13.7-17.6) Median bulb diam. 11.9 11.5 ± 1.2 (8.4-13.1) 12.8 ± 0.9 (10.9-14.6) Median bulb length/diam. 1.3 1.2 ± 0.1 (1.1-1.4) 1.2 ± 0.1 (1.1-1.4) Anterior end to excretory 71.4 67.6 ± 5.9 (57.1-77.0) 69.5 ± 9.9 (50.4-99.1) pore Anterior end to hemizonid 90.0 89.9 ± 8.3 (75.2-103.3) 97.0 ± 9.2 (86.5-116.8) Spicule (chord) 20.6 18.9 ± 1.4 (16.1-22.2) - Spicule (curved median - 19.1 17.7 ± 1.4 (14.6-19.8) line) Spicule (dorsal limb) 27.4 25.2 ± 1.8 (21.7-28.3) - Spicule (ventral limb) 13.0 11.6 ± 1.0 (9.6-13.5) - Ovary or testis length 450 383 ± 90.9 (212-580) 350 ± 63.1 (267-470) Post-uterine sac - - 77.2 ± 7.6 (60.8-87.7) Vulva diam - - 22.0 ± 1.8 (18.1-26.4) Vulva to anus distance - - 167 ± 16.4 (136-199) Post-uterine sac - - 46.2 ± 4.3 (37.5-53.7) length/Vulva to anus(%) Tail length 39.8 38.8 ± 3.3 (30.5-43.3) 43.4 ± 3.2 (37.4-48.3) Cloacal or anal body diam. 14.0 13.8 ± 1.4 (10.5-15.6) 11.3 ± 0.8 (9.5-12.7)

111

Fig. 6-3. Light photomicrographs of Aphelenchoides sp. 14Asp. A: Entire female and male; B: Head region; C, D: Lateral lines; E:Vulval region; F-H: Male tails; I-K: Female tails. (Scale bars = 20 μm.)

112

Fig. 6-4. Scanning electronic microscopy of Aphelenchoides sp. 14Asp. A: En face view of female head; B: Head region of female; C: Lateral view of female tail; D: Ventral view of female tail; E: Vulval region in ventral view; F: Vulval region in ventral view; G: Female tail; H: Head region of male; I: En face view of male head; J: Lateral Lines; K: Lateral view of male tail.

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides sp. 14Asp is characterized by body lengths of 634 (477-760) μm and 691 (617-771) μm for the male and female, respectively. The cuticle is weakly annulated and there are four incisures with equal width in the lateral field. The labial disc is clearly defined by a circular groove with hexagram-shaped outer labial sensillae. The stylet is 11 (10-11) μm with small basal swellings. The excretory pore is located between the nerve ring to anterior to the posteriomost margin of the

113 metacorpus or 68 (57-77) μm from the head. The spicules is convex curved, rose-thorn shaped with rounded apex and rostrum, dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip. The male tail bears six (2 + 2 + 2) caudal papillae ending in a pointed mucron which is without tiny nodular protuberances. The spermatheca is axial and oblong and contains disc-like sperm. The female tail is conoid or dorsally convex-conoid with hemispherical tail terminus step-like projection bearing tiny nodular protuberances in ventral. According to the categorization codes of OEPP/EPPO (2004), Aphelenchoides sp. 14Asp is defined as A2-B2-C1/3-D1-E1-F1/2.

Aphelenchoides sp. 14Asp has a female tail terminus bearing single mucron with tiny nodular protuberances. According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 2, which is defined as having the female tail terminus with “one or sometimes two mucronate structures”. Based on lateral line number, the stylet length and the female conoid or dorsally convex-conoid tail, it is close to eighteen species from Group 2, including A. absari Husain & Khan, 1967, A. arcticus Sanwal, 1965, A. brassicae Edward & Misra, 1969, A. echinocaudatus Haque, 1968, A. ensete Swart, Bogale & Tiedt, 2000, A. graminis Baranovskaya & Haque, 1968, A. haguei Maslen, 1979, A. macronucleatus Baranovskaya, 1963, A. nechaleos Hooper & Ibrahim, 1994, A. paranechaleos Hooper & Ibrahim, 1994, A. parasaprophilus Sanwal, 1965, A. richardsoni Grewal, Siddiqi & Atkey, 1992, A. singhi Das, 1960, A. suipingensis Feng & Li, 1986, A. tsalolikhini Ryss, 1993, A. tumulicaudatus Truskova, 1973, A. tuzeti B’Chir, 1979, A. xui Wang, Wang, Gu, Wang, and Li, 2013.

The new species differs from A. absari by the longer female body length = 683 (581-802) vs (390-450) μm, longer male body length = 634 (447-760) vs (320-430) μm, higher female ratio b = 10.5 (9.2-12.1) vs (4.0-4.5), higher male ratio b = 9.7 (7.1-11.9) vs (4.3-5.2), longer female tail length = 43.4 (37.4-48.3) vs 25 μm, longer male tail length = 38.8 (30.5-43.3) vs 28 μm, position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and longer spicule dorsal limb length = 25.2 (21.7-28.3) vs (15-19) μm. It differs from A. arcticus by longer spicule dorsal limb length = 25.2 (21.7-28.3) vs 21 μm, shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip) and male tail

114 terminus (bearing a pointed mucro without tiny nodular protuberances vs step-like projection). It differs from A. brassicae by the higher female ratio c’ = 3.9 (3.4-4.5) vs 2.75, longer female tail length = 43.4 (37.4-48.3) vs 21.5 μm, longer spicule dorsal limb length = 25.2 (21.7-28.3) vs (13-15) μm and shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip). It differs from A. echinocaudatus by the longer female body length = 683 (581-802) vs 427 μm, higher female ratio a = 28.8 (25.3-32.7) vs 24.5, higher female ratio b = 10.5 (9.2-12.1) vs 8, higher female ratio c’ = 3.9 (3.4-4.5) vs 2.7, longer female tail length = 43.4 (37.4-48.3) vs 22.8 μm, longer spicule dorsal limb length = 25.2 (21.7-28.3) vs (16-18) μm and shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip). It differs from A. ensete by the labial disc (clear defined with a circular groove vs clearly defined without a circular groove), lower female ratio a = 28.8 (25.3-32.7) vs 48.4 (36.9-61.8), lower male ratio a = 29.7 (27.3-32.7) vs 49.2 (41.7-58.1), longer spicule dorsal limb length = 25.2 (21.7-28.3) vs 18.2 (17.0-20.0) μm and shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip). It differs from A. graminis by the longer female body length = 683 (581-802) vs (390-560) μm, longer female tail length = 43.4 (37.4-48.3) vs 24.4 μm and longer spicule dorsal limb length = 25.2 (21.7-28.3) vs (15-19.2) μm and shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip). It differs from A. haguei by the position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip). It differs from A. macronucleatus by the position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and presences of male. It differs from A. nechaleos by the lower female ratio a = 28.8 (25.3-32.7) vs 41 (33-45), lower male ratio a = 29.7 (27.3-32.7) vs 41 (35-44), longer spicule dorsal limb length = 25.2 (21.7-28.3) vs 16 (15-20) μm and position of (P3) subventral postcloacal papillae (at mid of tail length vs at ca 65% of tail length posterior to cloacal slit). It differs from A. paranechaleos by the lower female ratio a

115

= 28.8 (25.3-32.7) vs 43 (37-46), lower male ratio a = 29.7 (27.3-32.7) vs 45 (40-50) and longer spicule dorsal limb length = 25.2 (21.7-28.3) vs 16 (15-18) μm. It differs from A. parasaprophilus by the shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip). It differs from A. richardsoni by the position of excretory pore (at same level of nerve ring or anterior to nerve ring vs posterior to nerve ring) and presences of male. It differs from A. singhi by the longer female body length = 683 (581-802) vs 490 μm, higher female ratio c’ = 3.9 (3.4-4.5) vs 2.3, longer female tail length = 46.2 (37.5-53.7) vs 28.5 μm, and longer spicule dorsal limb length = 25.2 (21.7-28.3) vs 17 μm. It differs from A. suipingensis by the shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards tip a tiny hook distal tip with slightly notched curvature near distal tip). It differs from A. tsalolikhini by the higher female ratio b = 10.5 (9.2-12.1) vs 6.7 (6.4-7.4), higher male ratio b = 9.7 (7.1-11.9) vs 5.8 (5.3-6.5), longer spicule dorsal limb length = 25.2 (21.7-28.3) vs (18-19) μm, longer spicule chord length = 18.9 (16.1-22.2) vs (14-15) μm, shape of spicule (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smoothly curved towards pointed distal tip) and absences of two pairs of dark-stained sclerotized cells on the dorsal side of vagina. It differs from A. tumulicaudatus by the higher female ratio b = 10.5 (9.2-12.1) vs (3.9-5.5) and lower male ratio a = 29.7 (27.3-32.7) vs 45 (40.7-42.9). It differs from A. tuzeti by higher female ratio c’ = 4.0 (3.3-4.7) vs (2.7-3.3), longer post-uterine sac occupying 46.2% (37.5–53.7%) distance between vulva to anus vs occupying less than one third of this distances and presence of male. It differs from A. xui by the spicule shape (dorsal limb smoothly curved towards pointed distal tip vs dorsal limb convex curved towards a tiny hook distal tip).

116

Aphelenchoides sp. 18Asp

(Figs 6-5, 6-6)

MEASUREMENTS

See Table 6-4.

DESCRIPTION

Male

Body slender, cylindrical, J-shaped when heat-relaxed. Cuticle with fine annulations. Lateral field with four incisures (i.e., three ridges with the narrower mid ridge). Lip region rounded in lateral view, slightly offset, with clear annulation, about twice as broad as high. SEM en face observation showing a six-lobed head with six equally sized and partial fused cephalic sectors. Labial disc clearly defined with a slightly depression by a circular groove. Oral aperture rounded, diam. ca 0.2 μm, surrounded by six slightly elevated inner labial sensilla. Outer labial sensilla obscure. Amphidial apertures oval, positioned dorsolaterally on edges of labial plate of lateral lips, four cephalic papillae at same level as amphidial apertures on subdorsal and subventral lips. Stylet 11.7-12.6 μm long, with conspicuous basal swellings, conus occupying ca 40% of its total length. Procorpus cylindrical, ca 2 body diam. long. Metacorpus (median bulb) strongly developed, almost spherical or oval, with centrally situated valves. Dorsal pharyngeal gland orifice opening into lumen of metacorpus ca one metacorpal valve length anterior to metacorpal valve. Pharyngo-intestinal junction immediately posterior to base of metacorpus. Nerve ring ca half body diam. length posterior to metacorpus. Pharyngeal gland lobe slender, ca 2-2.5 body diam. long, overlapping intestine dorsally. Excretory pore located at same level with nerve ring or anterior to nerve ring but posterior to base of metacorpus, occasionally slightly anterior to base of metacorpus. Hemizonid ca one body diam. posterior to excretory pore. Testis single, located on left side of intestine, outstretched or anteriorly reflexed in some individuals. Anterior part of testis containing developing spermatocytes in one single row, and then larger spermatocytes arranged in one single column. Posterior part of testis ca 4-5 body diam. long, forming vas deferens, containing several well developed sperm. Posterior end of vas deferens is fused with rectum to form a narrow cloacal tube. Lips of cloacal opening slightly protruding. Spicules separate, rose-thorn shaped with rounded apex and rostrum, dorsal limb convex curved towards a bifurcated distal tip

117 with notched curvature near distal tip, ventral limb convex curved and ending slightly before the dorsal limb, cucullus absent. Gubernaculum absent. Tail conoid, ca 3.5-4.5 cloacal body diam. long, possessing pointed tip bearing a pointed mucro ca 1.5–2.5 μm long with tiny nodular protuberances. Three pairs of subventral papillae present: one pair subventral precloacal papillae (P2) located slightly anterior to cloacal slit, one pair subventral postcloacal papillae (P3) located at ca 65% of tail length posterior to cloacal slit, and one tiny pair of papillae (P4) situated at base of tail terminus. Bursa absent.

Female

Body slender, cylindrical, slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region similar to male. Reproductive system single, ventrally located, including ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac. Ovary single, outstretched, anteriorly with double flexure in some individuals, developing oocytes arranged in single row occasionally in 2 rows, several well developed oocytes arranged in single row, a large elongated oocyte observed close to oviduct in some individuals. Oviduct short and connected with spermatheca, an axial and oval sac ca 1.5-2.5 body diam. long and filled with rounded sperm cells. Crustaformeria constructed of relatively large rounded cells. Uterus thick-walled, sometimes containing one developed egg. Vagina slightly inclined anteriorly to body axis. Vulva a simple slit in ventral view, without vulval membranes in ventral and lateral view, anterior and posterior lips slightly protruding. Post-uterine sac ca 4-5 vulval body diam. long, extending for ca 40-60% of vulval-anus distance, sometimes filled with large sperm. Anus distinct, a dome-shaped slit in ventral view. Tail conoid, ca 3-3.5 anal body diam. long. with hemispherical or truncate tail terminus bearing single mucron ca 1.0-2.0 µm long with tiny nodular protuberances in ventral.

118

Table 6-4. Morphometrics of Aphelenchoides sp. 18Asp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n - 20 20 L 884 816 ± 54.9 (655-912) 931 ± 66.7 (830-1116) a 35.8 32.8 ± 3.1 (26.2-37.2) 31.1 ± 3.2 (25.1-36.3) b 11.6 10.7 ± 0.8 (9.0-12.2) 10.8 ± 1.2 (8.3-13.4) b' 6.0 5.9 ± 0.5 (5.1-7.0) 6.0 ± 0.4 (4.8-6.8) c 18.8 17.6 ± 1.2 (14.2-19.0) 18.4 ± 1.2 (16.8-20.9) c' 2.5 2.7 ± 0.3 (2.2-3.3) 4.0 ± 0.4 (3.3-4.7) V or T 62.6 63.3 ± 11.8 (32.1-83.2) 70.4 ± 1.0 (68.2-70.4) Body diam. 23.4 25.1 ± 2.3 (21.0-29.1) 30.0 ± 3.3 (26.2-36.6) Stylet length 11.8 12.2 ± 0.3 (11.7-12.6) 12.5 ± 0.4 (11.5-13.0) Lip diam. 6.9 6.7 ± 0.5 (5.5-7.4) 7.2 ± 0.4 (6.0-7.5) Lip height 2.8 3.1 ± 0.3 (2.4-3.9) 3.2 ± 0.3 (2.7-3.8) Lip diam./height 2.5 2.2 ± 0.1 (1.9-2.5) 2.3 ± 0.2 (1.9-2.6) Median bulb length 15.2 15.6 ± 0.9 (13.9-17.3) 18.5 ± 1.3 (16.7-22.0) Median bulb diam. 12.2 11.8 ± 0.5 (10.9-12.6) 13.4 ± 0.8 (12.4-15.8) Median bulb 1.3 1.3 ± 0.1 (1.2-1.5) 1.4 ± 0.1 (1.2-1.7) length/diam. Anterior end to 77.4 80.7 ± 8.5 (66.6-103.2) 83.6 ± 7.2 (71.6-100.8) excretory pore Anterior end to 110.6 ± 11.5 (88.5-133.3) 104.0 103.4 ± 7.1 (91.9-124.3) hemizonid Spicule (chord) 22.2 21.5 ± 1.1 (18.4-23.3) - Spicule (curved median - 21.0 20.0 ± 1.4 (16.5-22.1) line) Spicule (dorsal limb) 29.3 28.2 ± 1.7 (24.1-31.4) - Spicule (ventral limb) 15.3 14.2 ± 0.9 (11.5-15.3) - Ovary or testis length 553 515 ± 96.5 (293-675) 406 ± 62.1 (309-546) Post-uterine sac - - 112.5 ± 12.6 (95.7-145.1) Vulva diam - - 26.2 ± 2.2 (23.3-30.8) Vulva to anus distance - - 224 ± 13.5 (202-255) Post-uterine sac length/ - - 50.4 ± 5.9 (40.4-62.4) vulva to anus (%) Tail length 47.1 46.6 ± 3.2 (40.9-54.6) 50.6 ± 5.0 (42.8-61.2) Cloacal or anal body 14.9 15.1 ± 1.0 (13.2-17.0) 12.7 ± 1.0 (11.0-15.1) diam.

119

Fig. 6-5. Light photomicrographs of Aphelenchoides sp. 18Asp. A: Entire female and male; B: Head region; C: Lateral lines; D: Vulval region; E-G: Male tails; H-J: Female tails. (Scale bar in figure A = 100 μm, scale bars in other figures= 20 μm)

120

Fig. 6-6. Scanning electronic microscopy of Aphelenchoides sp. 18Asp. A: En face view of female head; B: Head region of female; C: Lateral view of female tail; D: Ventral view of female tail; E: Vulval region in ventral view; F: Vulval region in lateral view; G: Female tail; H: Head region of male; I: En face view of male head; J: Lateral Lines; K: Male tail; L: Tip of spicules.

121

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides sp. 18Asp is characterized by body lengths of 816 (655-912) μm and 931 (830-1116) μm for the male and female, respectively. The cuticle is weakly annulated and there are four incisures with narrower mid ridge in the lateral field. The labial disc is clearly defined by a circular groove and depressed. The stylet is 12-13 μm long with small basal swellings. The excretory pore is located between the nerve ring to anterior to the posteriomost margin of the metacorpus or 81 (67-103) μm from the head. The spicules are convex curved, rose-thorn shaped with rounded apex and rostrum, dorsal limb curved towards a bifurcated distal tip with notched curvature near distal tip. The male tail bears six (2 + 2 + 2) caudal papillae ending in a pointed mucron which is with tiny nodular protuberances. The spermatheca is axial and oblong and contains disc-like sperm. The female tail is conoid with hemispherical or truncate tail terminus bearing single mucron with tiny nodular protuberances ventrally. According to the categorization codes of OEPP/EPPO (2004), Aphelenchoides sp. 18Asp is defined as A2-B2-C1-D1-E1-F1/2.

Aphelenchoides sp. 18Asp has a female tail terminus bearing single mucron with tiny nodular protuberances. According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 2 which is defined as having the female tail terminus with “one or sometimes two mucronate structures”. Based on lateral line number, the stylet length and the female conoid tail, it is close to fifteen species from Group 2, including A. absari Husain & Khan, 1967, A. daubichaensis Eroshenko, 1968, A. echinocaudatus Haque, 1968, A. ensete Swart, Bogale & Tiedt, 2000, A. graminis Baranovskaya & Haque, 1968, A. macronucleatus Baranovskaya, 1963, A. haguei Maslen, 1979, A. parasaprophilus Sanwal, 1965, A. richardsoni Grewal, Siddiqi & Atkey, 1992, A. singhi Das, 1960, A. suipingensis Feng & Li, 1986, A. tsalolikhini Ryss, 1993, A. tumulicaudatus Truskova, 1973, A. tuzeti B’Chir, 1979, A. xui Wang, Wang, Gu, Wang, and Li, 2013.

The new species differs from all these species by spicule shape (dorsal limb convex curved towards a bifurcated distal tip with notched curvature near distal tip vs dorsal limb convex or smoothly curved towards a pointed tip or clearly curved ventrally like a hook or male absent) and the position of (P3) subventral postcloacal papillae (at ca 65% of tail length posterior to cloacal slit vs at mid of tail length). Furthermore, it is also distinguished from A. absari Husain & Khan, 1967 by the longer female body

122 length = 931 (830-1116) vs (390-450) μm, longer male body length = 816 (655-912) vs (320-430) μm, higher female ratio b = 10.8 (8.3-13.4) vs (4.0-4.5), higher male ratio b = 10.7 (9.0-12.2) vs (4.3-5.2), longer female tail length = 50.6 (42.8-61.2) vs 25 μm, longer male tail length = 46.6 (40.9-54.6) vs 28 μm, position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and longer spicule dorsal limb length = 28.2 (24.1-31.4) vs (15-19) μm. It differs from A. daubichaensis the longer female body length = 931 (830-1116) vs (460-580) μm, longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 1.5 vulval body diam. long and presences of male. It differs from A. echinocaudatus by the longer female body length = 931 (830-1116) vs 427 μm, longer male body length = 816 (655-912) vs (365-475) μm, longer female tail length = 50.6 (42.8-61.2) vs 22.8 μm, longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 2 vulval body diam. long and longer spicule dorsal limb length = 28.2 (24.1-31.4) vs (16-18) μm. It differs A. ensete from by the labial disc (clear defined with a slightly depression by a circular groove vs clearly defined without a circular groove), longer female body length = 931 (830-1116) vs 635 (558-754) μm, lower female ratio a = 31.1 (25.1-36.3) vs 48.4 (36.9-61.8), lower male ratio a = 32.8 (26.2-37.2) vs 49.2 (41.7-58.1), longer male tail length = 46.6 (40.9-54.6) vs 31.6 (27.0-37.0) μm, longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 1-2 vulval body diam. long and longer spicule dorsal limb length = 28.2 (24.1-31.4) vs 18.2 (17.0-20.0) μm. It differs from A. graminis by the longer female body length = 931 (830-1116) vs (390-560) μm, longer female tail length = 50.6 (42.8-61.2) vs 24.4 μm and spicule dorsal limb length = 28.2 (24.1-31.4) vs (15-19.2) μm. It differs from A. haguei by the longer female body length = 931 (830-1116) vs 624 (560-765) μm and position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring). It differs from A. macronucleatus by the position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and presences of males. It differs from A. parasaprophilus by the longer female body length = 931 (830-1116) vs (680-750) μm and longer spicule dorsal limb length = 28.2 (24.1-31.4) vs 21 μm. It differs from A. richardsoni by the longer female body length = 931 (830-1116) vs (400-580) μm, position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring), longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 1-2 vulval body diam. and presences of male. It differs from A. singhi by the longer female body length = 931 (830-1116) vs 490 μm, longer male

123 body length = 816 (655-912) vs 510 μm, higher female ratio c’ = 4.0 (3.3-4.7) vs 2.3, longer female tail length = 50.6 (42.8-61.2) vs 28.5 μm and longer spicule dorsal limb length = 28.2 (24.1-31.4) vs 17 μm. It differs from A. suipingensis by the spicule shape (dorsal limb convex curved towards a bifurcated distal tip with notched curvature near distal tip vs dorsal limb clearly curved ventrally like a hook) and the position of (P3) subventral postcloacal papillae (at ca 65% of tail length posterior to cloacal slit vs at mid of tail length) which is mentioned above. It differs from A. tsalolikhini by the longer female body length = 931 (830-1116) vs 593 (546-640) μm, longer male body length, 816 (655-912) vs 555 (526-580) μm, longer spicule dorsal limb length = 28.2 (24.1-31.4) vs (18-19) μm, higher female ratio b = 10.8 (8.3-13.4) vs 6.7 (6.4-7.4), higher male ratio b = 10.7 (9.0-12.2) vs 5.8 (5.3-6.5), longer spicule chord length = 21.5 (18.4-23.3) vs (14-15) μm and absences of two pairs of dark-stained sclerotized cells on the dorsal side of vagina. It differs from A. tumulicaudatus by the longer female body length = 931 (830-1116) vs 593 (550-720) μm, higher female ratio b = 10.8 (8.3-13.4) vs (3.9-5.5), longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 2 vulval body diam. It differs from A. tuzeti by the longer female body length = 931 (830-1116) vs (340-690) μm, higher female ratio c’ = 4.0 (3.3-4.7) vs (2.7-3.3), longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 1-2.5 vulval body diam. and presences of male. It differs from A. xui by male ratio b = 10.7 (9.0-12.2) vs 7.7 (6.1-8.6) and outer labial sensilla (obscure vs hexagram).

124

Aphelenchoides sp. AspX

(Figs 6-7, 6-8)

MEASUREMENTS

See Table 6-5.

DESCRIPTION

Male Body slender, cylindrical, J-shaped when heat-relaxed. Cuticle with fine annulations. Lateral field with four incisures (i.e., three ridges of equal width). Lip region rounded in lateral view, slightly offset, with clear annulation, about twice as broad as high. SEM en face observation showing a six-lobed head with six equally sized and partial fused cephalic sectors. Labial disc clear defined with a circular groove. Oral aperture rounded, diam. ca 0.2 μm, surrounded by six slightly elevated inner labial sensilla. Outer labial sensilla hexagram-shaped. Amphidial apertures oval, positioned dorsolaterally on edges of labial plate of lateral lips, four cephalic papillae at same level as amphidial apertures on subdorsal and subventral lips. Stylet 9.9-11.3 μm long, with conspicuous basal swellings, conus occupying ca 40% of its total length. Procorpus cylindrical, ca 3 body diam. long. Metacorpus (median bulb) strongly developed, almost spherical or oval, with centrally situated valves. Dorsal pharyngeal gland orifice opening into lumen of metacorpus ca one metacorpal valve length anterior to metacorpal valve. Pharyngo-intestinal junction immediately posterior to base of metacorpus. Nerve ring ca half body diam. length posterior to metacorpus. Pharyngeal gland lobe slender, ca 2.5-3 body diam. long, overlapping intestine dorsally. Excretory pore located at same level with nerve ring or anterior to nerve ring but posterior to posteriomost margin of the metacorpus. Hemizonid ca one body diam. posterior to excretory pore. Testis single, located on left side of intestine, outstretched. Anterior part of testis containing developing spermatocytes in one single row, and then larger spermatocytes arranged in one single column. Posterior part of testis ca 4-5 body diam. long, forming vas deferens, containing several well developed sperm. Posterior end of vas deferens is fused with rectum to form a narrow cloacal tube. Lips of cloacal opening slightly protruding. Spicules separate, rose-thorn shaped with rounded apex, rostrum rounded or pointed, dorsal limb smooth curved towards a hook

125 distal tip with slightly notched curvature near distal tip, ventral limb convex curved and ending slightly before the dorsal limb, cucullus absent. Gubernaculum absent. Tail conoid, ca 3.5-5 cloacal body diam. long, possessing pointed tip bearing a mucro ca 2.0–3.0 μm long without tiny nodular protuberances. Three pairs of subventral papillae present: one pair subventral precloacal papillae (P2) located slightly anterior to cloacal slit, one pair subventral postcloacal papillae (P3) located at mid of the tail, one tiny pair of papillae (P4) situated at base of tail terminus. Bursa absent.

Female

Body slender, cylindrical, slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region similar to male. Reproductive system single single, ventrally located. Including ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac. Ovary single, outstretched, anteriorly with double flexure in some individuals, developing oocytes arranged in single row, several well developed oocytes arranged in single row, a large elongated oocyte observed close to oviduct in some individuals. Oviduct short and connected with spermatheca, an axial and oblong sac ca 2-3 body diam. long filled with disc-like sperm cells. Crustaformeria constructed of relatively large rounded cells. Uterus thick-walled, sometimes containing one developed egg. Vagina slightly inclined anteriorly to body axis. Vulva a simple slit in ventral view, without vulval membranes in ventral and lateral view, anterior and posterior lips slightly protruding. Post-uterine sac ca 3-5 vulval body diam. long, extending for ca 40-70% of vulval-anus distance, sometimes filled with large sperm. Anus distinct, a dome-shaped slit in ventral view. Tail conoid, ca 3.5-5 anal body diam. long. with hemispherical tail terminus with step-like projection ca 1.0-2.0 µm long with many tiny nodular protuberances in ventral.

126

Table 6-5. Morphometrics of Aphelenchoides sp. AspX. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n - 20 20 L 612 629 ± 48.0 (542-704) 691 ± 39.4 (617-771) a 29.4 31.8 ± 1.7 (28.4-34.6) 31.0 ± 1.6 (27.9-33.4) b 9.5 10.0 ± 0.6 (8.5-11.0) 10.2 ± 0.9 (8.8-11.9) b' 5.0 5.3 ± 0.6 (4.3-6.6) 5.7 ± 0.6 (4.6-7.0) c 18.3 16.9 ± 1.2 (15.3-19.0) 15.4 ± 1.4 (13.5-17.6) c' 1.8 2.2 ± 0.2 (1.7-2.7) 4.2 ± 0.5 (3.4-5.2) V or T 52.0 63.1 ± 9.9 (47.7-84.9) 66.6 ± 1.3 (64.3-68.6) Body diam. 20.8 19.8 ± 1.5 (16.8-22.7) 22.4 ± 2.1 (19.4-26.9) Stylet length 10.6 10.5 ± 0.4 (9.7-11.3) 10.6 ± 0.3 (9.9-11.3) Lip diam. 5.5 5.7 ± 0.3 (5.0-6.4) 5.9 ± 0.4 (5.0-6.6) Lip height 2.6 2.6 ± 0.1 (2.4-2.9) 2.7 ± 0.2 (2.2-3.1) Lip diam./height 2.0 2.1 ± 0.1 (1.9-2.4) 2.2 ± 0.1 (1.9-2.4) Median bulb length 12.5 12.6 ± 0.7 (11.0-14.0) 14.0 ± 1.0 (12.2-15.7) Median bulb diam. 10.4 10.2 ± 0.6 (8.8-11.2) 11.1 ± 0.8 (9.3-13.0) Median bulb length/diam. 1.3 1.2 ± 0.0 (1.1-1.3) 1.3 ± 0.1 (1.1-1.4) Anterior end to excretory 68.2 69.5 ± 5.8 (59.5-82.0) 73.3 ± 8.3 (58.2-90.4) pore Anterior end to hemizonid 83.2 93.2 ± 14.3 (72.3-119.5) 98.3 ± 9.1 (87.0-118.9) Spicule (chord) 16.8 18.5 ± 1.5 (16.3-21.7) - Spicule (curved median - 15.8 18.0 ± 1.5 (15.8-20.9) line) Spicule (dorsal limb) 21.5 24.3 ± 2.1 (21.4-28.4) - Spicule (ventral limb) 12.5 14.1 ± 1.3 (11.6-15.8) - Ovary or testis length 318 370 ± 51.0 (279-449) 290 ± 56.6 (191-406) Post-uterine sac - - 90.8 ± 17.1 (68.9-137.8) Vulva diam - - 19.9 ± 1.9 (17.1-24.0) Vulva to anus distance - - 185 ± 11.3 (168-220) Post-uterine sac length/ - - 48.7 ± 7.9 (39.1-69.5) vulva to anus (%) Tail length 33.5 37.5 ± 2.7 (32.3.2-42.9) 44.9 ± 3.5 (38.8-51.5) Cloacal or anal body diam. 12.5 12.9 ± 0.8 (11.4-14.0) 10.7 ± 1.5 (8.2-13.9)

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Fig.6-7. Light photomicrographs of Aphelenchoides sp. AspX. A: Entire female and male; B: Head region; C: Lateral lines；D: Vulval region；E-G: Male tails；H-J: Female tails. (Scale bar = 20 μm)

128

Fig. 6-8. Scanning electronic microscopy of Aphelenchoides sp. AspX. A: En face view of female head; B: Head region of female; C: Ventral view of female tail; D: Vulval region in ventral view; E, F: Female tail terminus; G: Head region of male; H: En face view of male head; I: Lateral Lines: J: Male tail.

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides sp. AspX is characterized by body lengths of 629 (542-704) μm and 691 (617-771) μm for the male and female, respectively. The cuticle is weakly annulated and there are four incisures with equal width in the lateral field. The labial disc is clearly defined by a circular groove with hexagram-shaped outer labial sensillae. The stylet is 11 (10-12) μm with small basal swellings. The excretory pore is located between the nerve ring to the posteriomost margin of the metacorpus or 68 (57-77) μm from the head. The spicules are smooth curved, rose-thorn shaped with

129 rounded apex and rostrum, dorsal limb smooth curved towards a hook distal tip with slightly notched curvature near distal tip. The male tail bears six (2 + 2 + 2) caudal papillae ending in a pointed mucron which is without tiny nodular protuberances. The spermatheca is axial and oblong and contains disc-like sperm. The female tail is conoid or dorsally convex-conoid with hemispherical tail terminus step-like projection bearing tiny nodular protuberances in ventral. According to the categorization codes of OEPP/EPPO (2004), Aphelenchoides sp. AspX is defined as A2-B2-C1-D1-E1-F1/2.

Aphelenchoides sp. AspX has a female tail terminus bearing single mucron with tiny nodular protuberances. According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 2 which is defined as having the female tail terminus with “one or sometimes two mucronate structures”. Based on lateral line number, the stylet length and the female conoid or dorsally convex-conoid tail, it is close to seventeen species from Group 2, including A. absari Husain & Khan, 1967, A. arcticus Sanwal, 1965, A. brassicae Edward & Misra, 1969, A. echinocaudatus Haque, 1968, A. ensete Swart, Bogale & Tiedt, 2000, A. graminis Baranovskaya & Haque, 1968, A. haguei Maslen, 1979, A. macronucleatus Baranovskaya, 1963, A. nechaleos Hooper & Ibrahim, 1994, A. paranechaleos Hooper & Ibrahim, 1994, A. parasaprophilus Sanwal, 1965, A. richardsoni Grewal, Siddiqi & Atkey, 1992, A. singhi Das, 1960, A. tsalolikhini Ryss, 1993, A. tumulicaudatus Truskova, 1973, A. tuzeti B’Chir, 1979, and A. xui Wang, Wang, Gu, Wang, and Li, 2013.

The new species differs from A. absari the longer female body length = 691 (617-771) vs (390-450) μm, longer male body length = 629 (542-704) vs (320-430) μm, higher female ratio b = 10.0 (8.5-11.0) vs (4.0-4.5), higher male ratio b = 10.2 (8.8-11.9) vs (4.3-5.2), longer female tail length = 44.9 (38.8-51.5) vs 25 μm, longer male tail length = 37.5 (32.3.2-42.9) vs 28 μm, position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and longer spicule dorsal limb length = 24.3 (21.4-28.4) vs (15-19) μm. It differs from A. arcticus by longer spicule dorsal limb length = 24.3 (21.4-28.4) vs 21 μm, shape of spicule (dorsal limb smooth curved towards a hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip) and male tail terminus (bearing a pointed mucro without tiny nodular protuberances vs step-like

130 projection). It differs from A. brassicae by the higher female ratio c’ = 4.2 (3.4-5.2) vs 2.75, longer female tail length = 44.9 (38.8-51.5) vs 21.5 μm, longer spicule dorsal limb length = 24.3 (21.4-28.4) vs (13-15) μm and shape of spicule (dorsal limb smooth curved towards a hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip). It differs from A. echinocaudatus by the longer female body length = 691 (617-771) vs 427 μm, higher female ratio a = 31.0 (27.9-33.4) vs 24.5, higher female ratio b = 10.0 (8.5-11.0) vs 8, higher female ratio c’ = 4.2 (3.4-5.2) vs 2.7, longer female tail length = 44.9 (38.8-51.5) vs 22.8 μm, longer spicule dorsal limb length = 24.3 (21.4-28.4) vs (16-18) μm and shape of spicule (dorsal limb smooth curved towards a hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip). It differs from A. ensete by the labial disc (clear defined with a circular groove vs clearly defined without a circular groove), lower female ratio a = 31.0 (27.9-33.4) vs 48.4 (36.9-61.8), lower male ratio a = 31.8 (28.4-34.6) vs 49.2 (41.7-58.1), longer spicule dorsal limb length = 24.3 (21.4-28.4) vs 18.2 (17.0-20.0) μm and shape of spicule (dorsal limb smooth curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip). It differs from A. graminis by the longer female body length = 691 (617-771) vs (390-560) μm, longer female tail length = 44.9 (38.8-51.5) vs 24.4 μm and longer spicule dorsal limb length = 24.3 (21.4-28.4) vs (15-19.2) μm and shape of spicule (dorsal limb smooth curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip). It differs from A. haguei by the position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and shape of spicule (dorsal limb smooth curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip). It differs from A. macronucleatus by the position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and presences of male. It differs from A. nechaleos by the lower male ratio a = 31.0 (27.9-33.4) vs 41 (35-44), longer spicule dorsal limb length = 24.3 (21.4-28.4) vs 16 (15-20) μm and position of (P3) subventral postcloacal papillae (at mid of tail length vs at ca 65% of tail length posterior to cloacal slit). It differs from A. paranechaleos by the lower female ratio a = 31.0 (27.9-33.4) vs 43 (37-46), lower male ratio a = 31.8 (28.4-34.6) vs 45 (40-50) and longer spicule dorsal limb length = 24.3 (21.4-28.4) vs 16 (15-18)

131

μm. It differs from A. parasaprophilus by the shape of spicule (dorsal limb smooth curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip). It differs from A. richardsoni by the position of excretory pore (at same level with nerve ring or anterior to nerve ring vs posterior to nerve ring) and presences of male. It differs from A. singhi by the longer female body length = 691 (617-771) vs 490 μm, higher female ratio c’ = 4.2 (3.4-5.2) vs 2.3, longer female tail length = 44.9 (38.8-51.5) vs 28.5 μm and longer spicule dorsal limb length = 24.3 (21.4-28.4) vs 17 μm. It differs from A. tsalolikhini by the higher female ratio b = 10.2 (8.8-11.9) vs 6.7 (6.4-7.4), higher male ratio b = 10.0 (8.5-11.0) vs (4.3-5.2) vs 5.8 (5.3-6.5), longer spicule dorsal limb length = 24.3 (21.4-28.4) vs (18-19) μm, longer spicule chord length = 18.5 (16.3-21.7) vs (14-15) μm, shape of spicule (dorsal limb smooth curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb smooth curved towards pointed distal tip) and absences of two pairs of dark-stained sclerotized cells on the dorsal side of vagina. It differs from A. tumulicaudatus by the higher female ratio b = 10.5 (9.2-12.1) vs (3.9-5.5) and lower male ratio a = 29.7 (27.3-32.7) vs 45 (40.7-42.9). It differs from A. tuzeti by higher female ratio c’ = 4.2 (3.4-5.2) vs (2.7-3.3), longer post-uterine sac occupying 48.7 (39.1-69.5) % of vulva to anus distance vs occupying less than one third of this distances, and presence of male. It differs from A. xui by the head region (no double groove on labial disc or lip sectors vs double groove on lip sectors).

132

Aphelenchoides sp. Asp25

(Figs 6-9, 6-10)

MEASUREMENTS

See Table 6-6.

DESCRIPTION

Male

Body slender, cylindrical, J-shaped when heat-relaxed. Cuticle with fine annulations. Lateral field with three incisures (i.e., two ridges). Lip region rounded in lateral view, slightly offset, with clear annulation, about twice as broad as high. SEM en face observation showing a six-lobed head with six equally sized and partial fused cephalic sectors. Labial disc present without a circular groove. Oral aperture rounded, diam. ca 0.2 μm, surrounded by six slightly elevated inner labial sensilla. Outer labial sensilla obscure hexagram shaped. Amphidial apertures oval, positioned dorsolaterally on edges of labial plate of lateral lips, four cephalic papillae at same level as amphidial apertures on subdorsal and subventral lips. Stylet 9.5-11.1 μm long, with conspicuous basal swellings, conus occupying ca 40% of its total length. Procorpus cylindrical, ca 1.5 body diam. long. Metacorpus (median bulb) strongly developed, almost spherical or pear-shaped, with centrally situated valves. Dorsal pharyngeal gland orifice opening into lumen of metacorpus ca one metacorpal valve length anterior to metacorpal valve. Pharyngo-intestinal junction immediately posterior to base of metacorpus. Nerve ring ca half body diam. length posterior to metacorpus. Pharyngeal gland lobe slender, ca 2-2.5 body diam. long, overlapping intestine dorsally. Excretory pore located at same level with nerve ring or anterior to nerve ring, but posterior to base of metacorpus. Hemizonid ca half body diam. posterior to excretory pore, but occasionally immediately posterior to excretory pore. Testis single, located on left side of intestine, outstretched or anteriorly reflexed in some individuals, developing spermatocytes in one single column. Anterior part of testis containing developing spermatocytes in single or double rows, and then larger spermatocytes arranged in one single column. Posterior part of testis ca 2-3 body diam. long, forming vas deferens, containing well developed sperm. Posterior end of vas deferens is fused with rectum to form a narrow cloacal tube. Lips of cloacal opening slightly protruding. Spicules

133 separate, smoothly arcuate, rose-thorn shaped with rounded apex and rostrum, condylus occasionally oblonged-rounded in some individuals, dorsal limb and ventral limb smoothly curved towards bluntly pointed distal tip, cucullus absent. Gubernaculum absent. Tail conoid, ca 2.5-3.5 cloacal body diam. long, possessing pointed tip bearing a short sharp mucro ca 0.5–1.5 μm long and without any tiny nodular protuberances. Three pairs of subventral papillae present: one pair subventral precloacal papillae (P2) located slightly anterior to cloacal slit, one pair subventral postcloacal papillae (P3) located at ca 60% of tail length posterior to cloacal slit, one tiny pair of papillae (P4) situated at base of tail terminus. Bursa absent.

Female

Body slender, cylindrical, slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region similar to male. Reproductive system single, ventrally located, including ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva and post-uterine sac. Ovary single, outstretched, anteriorly with double flexure in some individuals, developing oocytes arranged in 1-2 rows, several well developed oocytes arranged in single row, a large elongated oocyte observed close to oviduct in some individuals. Oviduct short and connected with spermatheca, an axial and oblong sac ca 2-3 body diam. long filled with disc-like sperm cells. Crustaformeria constructed of relatively large rounded cells. Uterus thick-walled, occasionally containing one developed egg. Vagina slightly inclined anteriorly to body axis. Vulva a simple slit in ventral view, without vulval flap in ventral and lateral view, anterior and posterior lips slightly protruding. Post-uterine sac ca 4-5 vulval body diam. long, extending for ca 35-50% of vulval-anus distance, sometimes filled with large sperm. Anus distinct, a dome-shaped slit in ventral view. Tail sub-cylindrical to cylindrical, ca 3-4 anal body diam. long. with hemispherical to obtusely truncate tail terminus bearing single mucron (>95% individuals) ca 0.5-1.5 µm long without any tiny nodular protuberances ventrally.

134

Table 6-6. Morphometrics of Aphelenchoides sp. Asp25. All measurements are in μm and in the form: mean ± s.d. (range).

Character Male Female Holotype Paratypes Paratypes n - 20 20 L 523 574 ± 57.3 (449-683) 711 ± 52.0 (603-822) a 28.8 26.9 ± 1.3 (24.8-29.6) 26.0 ± 1.6 (23.2-30.0) b 9.7 10.2 ± 0.7 (9.0-11.8) 11.9 ± 0.9 (10.4-13.4) b' 5.8 5.7 ± 0.3 (5.2-6.6) 6.3 ± 0.4 (5.6-7.1) c 17.5 16.7 ± 1.3 (14.0-19.1) 16.4 ± 1.4 (14.2-20.1) c' 2.6 2.7 ± 0.2 (2.3-3.3) 3.1 ± 0.2 (2.8-3.7) V or T 65.4 63.1 ± 9.9 (47.7-84.9) 69.5 ± 0.8 (68.2-71.3) Body diam. 18.2 21.4 ± 2.0 (17.2-23.8) 27.4 ± 2.7 (22.3-33.1) Stylet length 9.5 9.9 ± 0.3 (9.3-10.5) 10.4 ± 0.4 (9.5-11.1) Lip diam. 6.0 6.1 ± 0.3 (5.3-6.7) 6.5 ± 0.3 (5.9-7.3) Lip height 2.7 2.8± 0.1 (2.5-3.1) 2.9 ± 0.2 (2.6-3.4) Lip diam./height 2.3 2.2 ± 0.1 (2.0-2.5) 2.3 ± 0.1 (2.1-2.5) Median bulb length 11.4 11.5 ± 0.7 (10.1-12.6) 13.4 ± 0.9 (11.3-14.7) Median bulb diam. 9.4 10.3 ± 0.6 (9.0-11.0) 12.0 ± 0.7 (10.8-13.2) Median bulb length/diam. 1.2 1.1 ± 0.0 (1.0-1.2) 1.1 ± 0.1 (1.0-1.2) Anterior end to excretory 61.2 64.4 ± 5.6 (53.0-78.0) 70.1 ± 6.2 (58.2-82.5) pore Anterior end to hemizonid 77.5 82.5 ± 5.8 (70.5-92.1) 90.9 ± 6.9 (77.8-110.8) Spicule (chord) 15.6 16.5 ± 1.1 (14.1-18.8) - Spicule (curved median - 15.6 15.6 ± 1.1 (13.6-17.8) line) Spicule (dorsal limb) 20.2 21.5 ± 1.4 (18.7-23.8) - Spicule (ventral limb) 11.7 11.4 ± 1.0 (10.1-13.3) - Ovary or testis length 342 360 ± 71.1 (253-469) 319 ± 69.1 (234-450) Post-uterine sac - - 84.8 ± 12.5 (67.5-111.2) Vulva diam - - 23.0 ± 1.8 (20.1-27.1) Vulva to anus distance - - 181 ± 14.4 (150-214) Post-uterine sac length/ - - 47.6 ± 5.2 (35.4-48.3) vulva to anus (%) Tail length 29.9 34.6 ± 4.1 (27.5.2-44.0) 37.7 ± 1.7 (34.9-41.1) Cloacal or anal body diam. 11.3 12.7 ± 0.8 (10.5-14.1) 9.8 ± 0.7 (8.5-11.5)

135

Fig.6-9. Light photomicrographs of Aphelenchoides Asp25 n. sp. A: Entire female and male; B: Head region; C: Lateral lines; D: Vulval region; E, F: Male tails; G-I: Female tails. (Scale bar = 20 μm)

136

Fig. 6-10. Scanning electronic microscopy of Aphelenchoides sp. Asp25. A: En face view of female head; B: Head region of female; C: Female tail; D: Ventral view of female tail; E: Vulval region in lateral view; F: Female tail terminus; G: Head region of male; H: En face view of male head; I: Lateral Lines; J: Male tail.

DIAGNOSIS AND RELATIONSHIPS

Aphelenchoides sp. Asp25 is characterized by body lengths of 594 (449-683) μm and 711 (603-822) μm for the male and female, respectively. The cuticle is weakly annulated and there are three equal incisures in the lateral field. The labial disc is clear defined but without a circular groove. The stylet is 9-11 μm long with small basal swellings. The excretory pore is located between the nerve ring to posterior to the posteriomost margin of the metacorpus or 64 (53-78) μm from the head. The spicules smoothly arcuate, rose-thorn shaped with rounded apex and rostrum, dorsal limb and

137 ventral limb smoothly curved towards bluntly pointed distal tip. The male tail bears six (2 + 2 + 2) caudal papillae ending in a pointed mucron. The spermatheca is axial and oblong and contains disc-like sperm. The female tail is hemispherical to obtusely truncate with broadly rounded or cylindroid tail terminus bearing single mucron, which is without any tiny nodular protuberances in more than 95% of the individuals. According to the categorization codes of OEPP/EPPO (2004), Aphelenchoides sp. Asp25 is defined as A2-B2-C4-D1/2-E2-F1/2.

Aphelenchoides sp. Asp25 has a female tail terminus bearing single mucron without tiny nodular protuberances. According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 2 which is defined as having the female tail terminus with “one or sometimes two mucronate structures”. Based on the number of lateral lines and the stylet length, it is close to eleven species from Group 2, including A. agarici Seth & Sharma, 1986, A. chamelocephalus (Steiner, 1926) Filipjev, 1934, A. cibolensis Rifﬂe, 1970, A. eldaricus Esmaeili, Heydari, Golhasan, Kanzaki, 2017, A. indicus Chawla, Bhamburkar, Khan & Prasad, 1968, A. minoris Ebsary, 1991, A. parascalacaudatus Chawla, Bhamburkar, Khan & Prasad, 1968, A. rutgersi Hooper & Myers, 1971, A. sacchari Hooper, 1958, A. swarupi Seth & Sharma, 1986, and A. trivialis Franklin & Siddiqi, 1963.

The new species differs from Aphelenchoides agarici by shorter post-uterine sac occupying 47.6% (35.4–48.3%) of vulva-anus distance vs occupying more than half this distance. It differs from A. chamelocephalus by the smaller V value = 69.5 (68.2-71.3) vs (75-80) and the presence of post-uterine sac and male. It differs from A. cibolensis by the longer female body length = 711 (603-822) vs 410 (360-460) μm, longer female tail length = 37.7 (34.9-41.1) vs 26.6 μm, the presence of post-uterine sac and male. It differs from A. eldaricus by the higher b’ ratio of male = 5.7 (5.2-6.6) vs 3.8 (3.6-4.0) and female = 6.3 (5.6-7.1) vs 3.8 (3.0-4.9), higher T ratio = 63.1 (47.7-84.9) vs 33.1 (30.2-35.4), smaller male body diam. = 21.4 (17.2-23.8) vs 27 (26-28) μm, longer post-uterine sac = 84.8 (67.5-111.2) vs 29.8 (27.0-40.0) μm, smaller male cloacal body diam. = 12.7 (10.5-14.1) vs 19.0 (17.0-21.0) μm, smaller female anal body diam. = 9.8 (8.5-11.5) vs 15.7 (14.0-17.0) μm, smaller spicule size with = 21.5 (18.7-23.8) vs 40 (38-42) μm, 15.6 (13.6-17.8) vs 31 (29-33) μm, 11.4 (10.1-13.3) vs 27 (24-29) μm for the dorsal limb length, curved median line length and ventral limb length, respectively, and the female tail shape (sub-cylindrical to

138 cylindrical vs conical). It differs from A. indicus by the higher female b ratio = 11.9 (10.4-13.4) vs (9-10), lower female c’ ratio = 3.1 (2.8-3.7) vs 4.5, longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 3 vulval body diam. long, spicule dorsal limb length = 21.5 (18.7-23.8) vs 18 μm and female tail shape (sub-cylindrical to cylindrical with hemispherical to obtusely truncate tail terminus bearing a short single mucron in more than 95% individuals ca 0.5-1.5 µm long without any tiny nodular protuberances vs conoid with truncate terminus bearing a long sharply pointed mucro). It differs from A. minoris by the longer female body length = 711 (603-822) vs 413 (365-461) μm, higher female b ratio = 11.9 (10.4-13.4) vs 8.6 (8.1-9.0), higher female c’ ratio = 3.1 (2.8-3.7) vs 2.5, the longer female tail length = 37.7 (34.9-41.1) vs 23.8 μm, shorter post-uterine sac occupying 47.6% (35.4–48.3%) distance between vulva to anus vs occupying two third of this distances and tail shape (sub-cylindrical to cylindrical vs conoid and concave ventrally). Is differs from A. parascalacaudatus by the longer female body length = 711 (603-822) vs (400-500) μm, longer female tail length = 37.7 (34.9-41.1) vs 33.8 μm, position of excretory pore in of female (at same level with nerve ring to anterior to nerve ring but posterior to base of metacorpus vs posterior to nerve ring), female tail shape (sub-cylindrical to cylindrical vs conoid to dorsally convex-conoid) and presences of male. It differs from A. rutgersi by the longer female body length = 711 (603-822) vs (320-570) μm, longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 1-1.5 vulval body diam. long, longer female tail length = 37.7 (34.9-41.1) vs 30.5 μm, longer spicule dorsal limb length = 21.5 (18.7-23.8) vs (14-17) μm and position of (P3) postcloacal papillae (at ca 60% of tail length posterior to cloacal slit vs at mid of tail length posterior to cloacal slit). It differs from A. sacchari by the shorter female stylet length = 10.4 (9.5-11.1) vs 11.4 μm, longer spicule dorsal limb length = 21.5 (18.7-23.8) vs (15-17) μm and position of (P3) postcloacal papillae (at ca 60% of tail length posterior to cloacal slit vs at mid of tail length). It differs from A. swarupi by the spicule shape (dorsal limb smoothly curved towards bluntly pointed distal tip vs dorsal limb convex curved and a notched curvature near distal end) and position of (P3) postcloacal papillae (at ca 60% of tail length posterior to cloacal slit vs at mid of tail length). It differs from A. trivialis by longer female body length = 711 (603-822) vs 431(369-397) μm, higher female c’ ratio = 3.1 (2.8-3.7) vs 2.1, longer female tail length = 37.7 (34.9-41.1) vs 23.3 μm, longer post-uterine sac occupying ca 4-5 vulval body diam. long vs ca 1 vulval body diam. long, the female tail shape (sub-cylindrical to cylindrical vs

139 dorsally convex-conoid) and the presences of spermatheca and male.

MOLECULAR PROFILES AND PHYLOGENETIC STATUS

The sequences of six populations for the partial 18S and 28S D2-D3 were generated and deposited in GenBank, the information is given in Table 6-7. Alignment of the sequences with MAFFT resulted in datasets of 1914 characters for 18S and 1045 characters for 28S D2-D3. Phylogenetic relationships among the isolates were determined separately for each dataset using Bayesian inference (BI) with Aphelenchus avenae as outgroup. The 50% majority rule consensus phylogenetic trees were generated from 18S dataset alignment by BI analysis under the TIM3 + I + G model, and from 28S D2-D3 under TIM2 + I + G model.

Table 6-7. The information of sequences of six Aphelenchoides populations

18S 28S D2-D3 Species Code length number length number

1638bp KY689016 763bp KX356748 Aphelenchoides sp. 12Asp AspSDCD 1672bp KY769068 710bp KY769083 Asp6266 1673bp KY689018 775bp KY689009 Aphelenchoides sp. 14Asp AspHK 1657bp KY769058 773bp KY769072 Aphelenchoides sp. 18Asp AspJSYZ 1657bp KY769061 771bp KY769076 Aphelenchoides sp. AspX AspJSYX Aphelenchoides sp. Asp25 AspSDSG 1708bp KY769062 782bp KY769077

In the phylogenetic tree based on sequences of 18S (Fig. 6-11), Aphelenchoides sp. 12Asp, Aphelenchoides sp. Asp6266 and A. macronucleatus comprised a single clade with maximal posterior probability support (100) , and this single clade is sister to an unidentified species Aphelenchoides sp. JB012. The comparison of the pairwise sequence similarities between Aphelenchoides sp. 12Asp, Aphelenchoides sp. Asp6266 and A. macronucleatus revealed 99.81% and 99.43% similarity value, respectively, which strongly indicates that these three population are conspecific. Morphologically, Aphelenchoides sp. 12Asp and A. macronucleatus are very close but only different in the presence of males. The 18S sequence of Aphelenchoides sp. 12Asp is 92.17% similar to an unidentified species Aphelenchoides sp. JB012.

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Aphelenchoides sp. 14Asp and Aphelenchoides sp. 18Asp clustered with A. xui and 12 unidentified Aphelenchoides species into a maximally supported clade (PP= 100). The pairwise sequence similarities between these two new species and other populations in this clade were given in the matrix (Fig. 6-12.). Aphelenchoides sp. 14Asp shared the high similarity value with 98.98%, 98.76%, 98.86%, 99.34%, 98.98%, 98.39% and 98.39% with Aphelenchoides sp. 13Asp, Aphelenchoides sp. 15Asp, Aphelenchoides sp. 16Asp, Aphelenchoides sp. 17Asp, Aphelenchoides sp. 19Asp, Aphelenchoides sp. 7763Asp and Aphelenchoides sp. 8Asp, respectively. This together with the fact they are in the same clade indicates that these 7 population are conspecific. The similarity value between Aphelenchoides xui, Aphelenchoides sp. 18Asp, Aphelenchoides sp. Asp2241, Aphelenchoides sp. Ap_A, Aphelenchoides sp. SAS_2006, and Aphelenchoides sp. YN which are also clustered in the maximally supported larger clade is 93.58%, 94.92%, 95.69%, 96.90%, 93.55% and 93.31%, respectively. Morphologically, the differences between Aphelenchoides sp. 14Asp and A. xui are mentioned above. Aphelenchoides sp. 14Asp can be easily distinguished from Aphelenchoides sp. 18Asp by the spicule shape (dorsal limb convex curved towards a tiny hook distal tip with slightly notched curvature near distal tip vs dorsal limb convex curved towards a bifurcated distal tip with notched curvature near distal tip). However, no morphology data of Aphelenchoides sp. Asp2241, Aphelenchoides sp. Ap_A, Aphelenchoides sp. SAS_2006, and Aphelenchoides sp. YN are available.

The sequence of Aphelenchoides sp. 18Asp is only 94.38%, 94.92%, 94.48%, 94.32%, 94.50%, 94.44%, 93.78%, 93.84%, 94.56%, 94.77%, 92.87%, 92.40% and 93.58% similar to Aphelenchoides sp. 13Asp, Aphelenchoides sp. 14Asp, Aphelenchoides sp. 15Asp, Aphelenchoides sp. 16Asp, Aphelenchoides sp. 17Asp, Aphelenchoides sp. 19Asp, Aphelenchoides sp. Asp2241, Aphelenchoides sp. 7763Asp, Aphelenchoides sp. Asp8, Aphelenchoides sp. Ap_A, Aphelenchoides sp. SAS_2006, Aphelenchoides sp. YN and Aphelenchoides xui, respectively. Morphologically, the unique spicule shape, i.e., dorsal limb convex curved towards a bifurcated distal tip with notched curvature near distal tip, is clearly distinctive from Aphelenchoides sp. 14Asp and Aphelenchoides xui.

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Fig. 6-11. Phylogenetic relationships of 6 Aphelenchoides populations and aphelenchid nematodes based on near full length of 18S. The 10 001st Bayesian tree inferred from 18S under TIM2 + I + G model (ln L = - 34980.5023; freqA = 0.1902; freqC = 0.1853; freqG = 0.3095; freqT = 0.3150; R(a) = 1.2931; R(b) = 3.6886; R(c) = 1.2931; R(d) = 1.0000; R(e) = 4.5596; R(f) = 1.0000; Pinvar = 0.0610; Shape = 0.9030). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

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Aphelenchoides sp. AspX clustered with 6 unidentified species into a weakly supported clade (PP: 78) clade. The pairwise sequence similarities between Aphelenchoides sp. AspX and Aphelenchoides sp. Ap007, Aphelenchoides sp. Ap052, Aphelenchoides sp. Ap055, Aphelenchoides sp. Ap108, Aphelenchoides sp. Ap110 and Aphelenchoides sp. G001 are only 93.66%, 91.75%, 95.62%, 90.95%, 92.81% and 93.12%, respectively. Morphological data of these species are not available.

Aphelenchoides sp. Asp25 is in an unresolved position together with 4 Aphelenchoides bicaudatus populations, 5 unidentified Aphelenchoides species into a poorly supported clade (PP: 50). The sequence of Aphelenchoides sp. Asp25 is only 90.95% to 92.47% similar with these 4 Aphelenchoides bicaudatus populations. Morphologically, Aphelenchoides sp. Asp25 differs from Aphelenchoides bicaudatus by the number of lateral lines (3 vs 2) and the female tail shape (tail sub-cylindrical to cylindrical with hemispherical to obtusely truncate tail terminus bearing single mucron without any tiny nodular protuberances in ventral vs tail conoid with a bifurcate mucron). The new species is molecularly also clearly different from two unknown species, only 92.78% (Aphelenchoides sp. Asp1764) and 92.78% (Aphelenchoides sp. Asp3912) similar, respectively. These two unknown species are highly similar (99.76%) which may indicate that they are conspecific. Furthermore, these two populations has the same type at female tail shape i.e., sub-cylindrical to cylindrical with hemispherical to obtusely truncate tail terminus bearing single mucron, which is with tiny nodular and like a brush. The female tail shape of Aphelenchoides sp. Asp25 is sub-cylindrical to cylindrical with hemispherical to obtusely truncate tail terminus bearing a single mucron without any tiny nodular protuberances. This character can be used to distinguishing them. The new species is also only 92.61% (Aphelenchoides sp. 10Asp), 92.25% (Aphelenchoides sp. K1_WY_2008) and 93.06% (Aphelenchoides sp. JB011), similar to these undescribed species. However, the latter three unknown species clustered into an clade with high posterior probability (100) and short tree branch shared which may indicate that they are conspecific. Morphologically, Aphelenchoides sp. Asp25 is very closed to Aphelenchoides sp. K1_WY_2008, the only difference is the head region shape (labial disc present without a circular groove vs labial disc present with a circular groove).

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Fig. 6-12. Sequences similarity of 18S between Aphelenchoides 14Asp n. sp. and Aphelenchoides 14Asp n. sp. with other 13 Aphelenchoides spp. in same clade, the species with high pairwise sequences similarity value were marked by red (>98%).

In the phylogenetic tree based on sequences of 28S D2-D3 region (Fig. 6-13.), Aphelenchoides sp. 12Asp and Aphelenchoides sp. Asp6266, Aphelenchoides fragariae isolate CA21_USA and an unidentified species Aphelenchoides sp. 11Asp clustered into a maximally supported clade clade. Aphelenchoides sp. 12Asp and Aphelenchoides sp. Asp6266 are maximally supported together in one clade and both sequences are highly similar (99.01%) which also strongly indicate that they are conspecific. Aphelenchoides sp. 12Asp is molecularly very different from Aphelenchoides fragariae isolate CA21_USA and Aphelenchoides sp. 11Asp, 80.46% and 84.41%, similarity respectively. Aphelenchoides sp. 12Asp can also be clearly differentiated from Aphelenchoides fragariae in morphology by the lateral lines number (4 or 6 vs 2). However, no morphology data of Aphelenchoides sp. 11Asp are available.

Aphelenchoides sp. 14Asp and Aphelenchoides sp. 18Asp clustered with Aphelenchoides xui and 14 unidentified Aphelenchoides species into a maximally supported clade. The pairwise sequence similarities between these two new species and other population in this clade are provided in the matrix (Fig. 6-14). Aphelenchoides sp. 14Asp is relatively moderate to relatively highly similar, 93.68%, 98.84%, 95.23%, 98.19%, 96.39%, 98.97%, 98.58%, 93.54%, 92.39% and 94.74% with Aphelenchoides sp. 13Asp, Aphelenchoides sp. 15Asp, Aphelenchoides sp. 16Asp, Aphelenchoides sp. 17Asp, Aphelenchoides sp. 19Asp, Aphelenchoides sp. 20Asp, Aphelenchoides sp. 9Asp, Aphelenchoides sp. 7763Asp, Aphelenchoides sp. 8Asp and Aphelenchoides sp. 8503-2, respectively, all belonging to a maximally supported clade.

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Aphelenchoides sp. 18Asp has a maximally supported sister relationship with the group discussed above. Similarity values were provided in Fig. 6-14 and these range from 77.87% to 85.62%. Its sister position and high molecular differences indicate this species is a new species.

Aphelenchoides sp. AspX clustered with Aphelenchoides rotundicaudatus and 16 unidentified species into low posterior probability supported (74) clade. The pairwise sequence similarities between Aphelenchoides sp. AspX and other species in this clade ranged from 67.63% to 83.41%, no morphological data of these species are available except for Aphelenchoides rotundicaudatus. Aphelenchoides sp. AspX is differs from Aphelenchoides rotundicaudatus by the male tail shape (tail conoid possessing pointed tip bearing a mucro without tiny nodular protuberances vs tail cylindrical, terminus usually broad rounded, occasionally bearing a short and blunt cuticular thickening or peg) and female tail shape (tail conoid with hemispherical tail terminus with step-like projection long with many tiny nodular protuberances in ventral vs tail cylindrical terminus broadly rounded with a very small cuticular thickening or bearing a blunt peg).

Aphelenchoides sp. Asp25 is sister to Aphelenchoides stellatus (98.98% similar) and clusters with Aphelenchoides sp. Asp1764 (, Aphelenchoides sp. CA22 and Aphelenchoides sp. K1_WY_2008 into a maximally supported clade. However, morphologically, Aphelenchoides sp. Asp25 differs from Aphelenchoides stellatus by the lateral lines number (3 vs 4), male tail shape (tail conoid possessing pointed tip bearing a short sharp mucro without any tiny nodular protuberances vs tail conical, terminus bearing a star-like peg) and female tail shape (tail sub-cylindrical to cylindrical with hemispherical to obtusely truncate tail terminus bearing single mucron without any tiny nodular protuberances in ventral vs tail elongate conoid, slightly ventrally arcuate, terminus armed with a star-like tetramucronate process). Aphelenchoides sp. Asp25 is molecular also clearly different from the following closely related species, 80.90%, 80.39% and 82.40% similar to Aphelenchoides sp. Asp1764, Aphelenchoides sp. CA22 and Aphelenchoides sp. K1_WY_2008, respectively. The status of Aphelenchoides sp. Asp25 with other species in 28S D2-D3 tree is also consistent with its position in the phylogenetic tree based on sequences of 18S.

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Fig. 6-13. Phylogenetic relationships of 6 Aphelenchoides populations and aphelenchid nematodes based on near full length of 18S. The 10 001st Bayesian tree inferred from 28S D2-D3 under TIM2 + I + G model (ln L = - 34980.5023; freqA = 0.1902; freqC = 0.1853; freqG = 0.3095; freqT = 0.3150; R(a) = 1.2931; R(b) = 3.6886; R(c) = 1.2931; R(d) = 1.0000; R(e) = 4.5596; R(f) = 1.0000; Pinvar = 0.0610; Shape = 0.9030). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades

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Fig. 6-14. Sequences similarity of 28S D2-D3 between Aphelenchoides 14Asp n. sp. and Aphelenchoides 14Asp n. sp. with other 15 Aphelenchoides spp. in same clade. The species with high pairwise sequences similarity value were marked by red (>98%).

Thus, molecular profiles and phylogenetic analyses strongly supported the status of Aphelenchoides sp. 12Asp, Aphelenchoides sp. 14Asp, Aphelenchoides sp. 18Asp, Aphelenchoides sp. AspX and Aphelenchoides sp. Asp25 as new species.

Discussion

The genus Aphelenchoides has few morphologically diagnostic taxonomic characters, high morphological variability and the morphometrically values often overlap, even if the species are very detailed described based on a comprehensive morphological analysis. Therefore, it is difficult to compare putative new species with old descriptions (Hockland 2001, Kanzaki 2006). Hockland (2001) evaluated the discriminatory power of the diagnostic characters, which are frequently used to identify Aphelenchoides species while testing her proposed polytomous key. Briefly, tail terminus shape, post-vulval uterine sac length and lateral incisures number were considered to have a relatively high discriminating power. However the tail terminus shape can be variable in some species (Hooper & Myers, 1971) and the lateral lines could also be variable, for example 4 or 6 lateral lines can often only reliably investigated under SEM (Hooper & Ibrahim, 1994).

Furthermore, these “high discriminating power” characters or other morphologically diagnostic taxonomic characters did not distinguish Aphelenchoides sp. Asp25 and Aphelenchoides sp. KY_2008, Aphelenchoides sp. 14Asp and Aphelenchoides sp. AspX. Head region characters of Aphelenchoides are efficient to discriminate some close species such as A. besseyi and A. fujianensis, A. ritzemabosi and A. gorganensis (see Chapter VII). Only head region characters allow to differentiate between

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Aphelenchoides sp. Asp25 and Aphelenchoides sp. KY_2008 (labial disc present without a circular groove vs labial disc present with a circular groove), Aphelenchoides sp. 14Asp and Aphelenchoides sp. AspX (double groove on lip sector vs no double groove on labial disc or lip sectors). This finding highlights the potential of head region characters to discriminate Aphelenchoides species. The spicule condylus were recommended by Hockland (2001) to identify Aphelenchoides species that were used to distinguish A. besseyi and A. fujianensis (Jesus et al., 2016). Furthermore, the distal tip of the spicule is also an important character for Aphelenchoides sp. 18Asp, i.e. a dorsal limb convex curved towards a bifurcated distal tip is unique within Aphelenchoides. In A. xui , A. fuchsi and A. paraxui, the spicule dorsal limb with a hook-like tip were noticed to be an important character (Wang et al., 2013, Esmaeili et al. 2016b, 2017b). The curvature of the spicule is an often overlooked character which is only used to separate A. nechaleos and A. paranechaleos (Hooper & Ibrahim, 1994).

Currently, species discrimination in Aphelenchoides are mainly based on molecular profiles. The obtained tree topologies the 18S and 28S D2-D3 agree with recent studies that divide Aphelenchoides species into two major clades, and the fact that several closely related genera are embedded within Aphelenchoides sequences supporting the paraphyly of this genus (Sánchez-Monge et al., 2016; Esmaeili et al., 2016b, Kanzaki et al., 2014c, Kanzaki & Giblin-Davis 2012, Rybarczyk-Mydłowska et al., 2012; Jesus et al., 2016). Despite that recent descriptions of Aphelenchoides are more accurate and include both, morphology and molecular data (Wang et al., 2013, Fang et al., 2014a, b; Esmaeili et al., 2016a, b, c; Jesus et al., 2016; Sánchez-Monge et al., 2017) clade 2a still shows a complex polyphyletic relationship. Moreover, Aphelenchoides, Laimaphelenchus and Schistonchus s.l. still cannot be clearly separated, possibly implying that all three genera share a recent common ancestor (Zeng et al., 2007).

Remarkably, some phylogenetic positions are different between the in 18S and 28S D2-D3 tree. For example, A. huntensis is in clade 2a in the 18S tree but outside this clade in the 28S D2-D3 trees; also A. fuchsi clusters with A. iranicus in an early branch of clade 2a in the 18S but clusters with a unidentified species Aphelenchoides sp. BE1 into a group in clade 2a. However, Aphelenchoides sp. 14Asp and other populations which are mentioned above (in Fig. 6-12 and Fig. 6-14) clustered into a

148 highly supported clade in clade 2a, showing a high consistency of 18S and 28S D2-D3 and agreement of molecular and morphological data. Studies on the intra-specific variation are essential in order to assess characters for their degree of constancy and stability (Hockland 2001). The populations in clade 2a need further research, including more detailed observations because several morphology characters can be only seen under SEM. The combination of more observations of LM in combination with SEM and molecular tools, will shed new light on the intra-specific variation and inter-specific variation of this genus.

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Chapter VII

Mapping head region characteristics on the molecular phylogeny of Aphelenchoididae

Manuscript in preparation:

Yiwu FANG1,2, Alcides SÁNCHEZ-MONGE2,3, Marjolein COUVREUR2, Qing Xue2, Hongmei

LI1, Wim BERT2

1 Department of Plant Pathology, Nanjing Agricultural University, Nanjing

210095, P.R. China

2 Ghent University, Department of Biology, Nematology Research Unit,

Ledeganckstraat 35, B-9000 Ghent, Belgium.

3 Universidad de Costa Rica, Laboratorio de Nematología, Centro de Investigación en

Protección de Cultivos (CIPROC), 2060, Costa Rica

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Abstract

Aphelenchoididae Skarbilovich, 1947 includes an ecologically and morphologically diverse array of species. The results of concatenated phylogenetic analyses combining 18S rDNA and D2-D3 28S rDNA gene sequences from 404 taxa revealed strong support for 5 major clades of Aphelenchoididae. However, some morphologically well defined genera are surprisingly paraphyletic or polyphyletic.. In order to clarify the taxonomic status of the different genera in Aphelenchoididae, we studied the detailed morphology of the head region, which is often neglected in other studies. The ultrastructure study of head region of 78 species from 11 genera resulted in 40 head region types, based on the oral apertures, inner labial sensilla, outer labial sensilla, cephalic sensilla, amphids apertures and annulations. The head region types are efficient to distinguish several closed species, such as Aphelenchoides besseyi and A. fujianensis. However, in general the type of head region does not correspond to the molecularly based relationships. Further work combing both molecular and ultrastructural data is still needed in Aphelenchoididae to understand this complex taxon.

Keywords: Aphelenchoididae, concatenated analysis, molecular phylogeny, ribosomal DNA, scanning electron microscopy, head region, morphology, taxonomy.

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Introduction

The family Aphelenchoididae Skarbilovich, 1947 is established as a large group of stylet-bearing nematodes adapted to a wide range of ecological relationships including predation, fungus feeding, facultative plant-parasitism and obligate insect parasitism (Nickle, 1970). The family Aphelenchoididae comprises 6 subfamilies and 25 genera according to Hunt (2008) and two more genera, Devibursaphelenchus and Pseudaphelenchus Kanzaki & Giblin-Davis, 2009 were added recently. For the economically important genus Aphelenchoides Fischer, 1894, 14 plant-parasitic species have been reported causing damage to a broad range of plants and crops (McCuiston et al., 2007, Rybarczyk-Mydłowska et al., 2012, Jones et al., 2013, Sánchez-Monge et al., 2015). Another important genus; Bursaphelenchus Fuchs, 1937; has received ample attention due to the economical importance of the pine-wilt nematode i.e., B. xylophilus (Steiner & Buhrer, 1934) Nickle,1970 (Futai, 2013). This genus is well described and their intrageneric relationships are supported by molecular phylogenies and morphological data (Ryss et al., 2005, Ye et al., 2007; Braasch et al., 2009). However, the phylogenetic complexity of Aphelenchoididae genera, except Bursaphelenchus, was clearly revealed by analyzing the 18S and 28S ribosomal DNA (rDNA) sequences (Kanzaki & Davis-Giblin 2012, Ryss et al., 2013，Sánchez-Monge, 2016).

The morphological characters including spicule, stylet, bursa, tail tip shape, position of the excretory pore, body shape and size of female, can be used to differentiate most genera of the family (Nickle 1970). However, some genera, i.e., Aphelenchoides and Laimaphelenchus, lack diagnostic features and overlap in several characters (Davies et al., 2015). The phylogeny based on 18S and 28S rDNA also revealed the paraphyletic statuses of Aphelenchoides and Laimaphelenchus (Rybarczyk-Mydłowska et al., 2012, Ryss et al., 2013, Fang et al., 2014, Sánchez-Monge et al., 2016, Esmaeil et al., 2016). Another example is the complex phylogeny of Schistonchus Cobb, 1927. Davies et al. (2015) clarified and segregated Schistonchus s. l. into three separate genera namely Ficophagus n. gen, Martininema n. gen and Schistonchus s. s., based on the taxonomy, phylogeny, distribution and co-evolution information, and especially traits related to the head region.

However, since the availability of comprehensive data related to the head region is

153 limited for most genera, it is unknown to which extent this is informative to us this to diagnose the genera and understand their phylogenetic relations. In this paper we analysed the head region traits of Aphelenchoididae, revealed by Scanning Electron Microscopy, on a molecular framework based on two ribosomal genes, i.e., 18S and 28S, in order to shed new light on the phylogeny and generic delimitations within Aphelenchoididae

Materials and methods

NEMATODE CULTURING AND MORPHOLOGICAL OBSERVATIONS

Nematode samples were collected from different habitats in geographical widespread localities (Table. 7-1) and were isolated using the modified Baermann funnel technique for 24h. Several populations were multiplied successfully onto Botryotinia fuckeliana (de Bary) Whetz. growing on potato dextrose agar cultures for 2-4 weeks at 25°C. Measurements were made from the fungal culture specimens fixed in TAF and processed to glycerin following the method of Seinhorst (1959). Light micrographs were made using an Olympus BX51 microscope equipped with an Olympus DP27 camera. However, the diagnosis and description of unknown taxa was out of the scope of this paper, therefore only descriptive code names were used as reference for newly generated sequences (see Table 7-1).

Table 7-1. List of Aphelenchoides populations which were provided in this study. The populations with SEM in this study are in bold.

Reference code Origin Substrate Culture GenBank /host accession number 18S 28S Aphelenchoides besseyi A030 Costa Rica Oryza sativa No KX356700 KX356760 A. besseyi A003 Costa Rica Phaseolus No KX356697 KX356753 vulgaris Aphelenchoides sp. 12Asp China Pinus thunbergii Yes KY689016 KX356748 Aphelenchoides sp. 18Asp China Oryza sativa Yes KY769058 KY769072 Aphelenchoides sp. AspX China Pinus Yes KY769061 KY769076 massoniana Aphelenchoides sp. Asp25 China Lycopersicon Yes KY769062 KY769077 esculentum A. fujianensis Asp4993 USA Wood No KY769067 KY769082 packaging A. unisexus 4Asp Germany Wood Yes KY689010 - packaging A. unisexus 5Asp South Korea Unknown Yes KY689011 - A. unisexus 6Asp Australia Wood Yes KY689012 KY689005 packaging

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A. unisexus Asp86 China Pinus Yes KY769060 KY769075 massoniana Aphelenchoides sp. 8Asp China Ipomoea Yes KY689013 KY689006 batatas Aphelenchoides sp. 9Asp USA Juniperus Yes - KY689007 virginiana Aphelenchoides sp. 10Asp China Solanum Yes KY689014 - tuberosum Aphelenchoides sp. 11Asp China Pinus Yes KY689015 KY689008 massoniana Aphelenchoides sp. 13Asp Taiwan Wood Yes KY689017 KX356749 packaging Aphelenchoides sp. 14Asp Hongkong Wood Yes KY689018 KY689009 packaging Aphelenchoides sp. 15Asp Taiwan Apium graveole Yes KY689019 KX356750 ns Aphelenchoides sp. 16Asp Brazil Wood Yes KY689020 KX356751 packaging Aphelenchoides sp. 17Asp South Africa Wood Yes KY689021 KX356752 packaging Aphelenchoides sp. 19Asp Taiwan Wood Yes KY769059 KY769073 packaging Aphelenchoides sp. Asp1764 USA Sabina No KY769063 KY769078 virginiana Aphelenchoides sp. Asp2241 Unknown Wood No KY769064 KY769079 packaging Aphelenchoides sp. Asp2890 South Korea Wood No - KY769080 packaging Aphelenchoides sp. Asp3187 Unknown Wood No KY769065 KY769081 packaging Aphelenchoides sp. Asp3912 Japan Podocarpus No KY769066 - macrophyllus Aphelenchoides sp. Asp6266 Japan Acer palmatum No KY769068 KY769083 Aphelenchoides sp. Asp6753 United Wood No KY769069 KY769084 Kingdom packaging Aphelenchoides sp. Asp7763 South Korea Wood Yes KY769070 KY769085 packaging Aphelenchoides sp. Asp75747 Germany Wood No KY769071 KY769086 packaging Aphelenchoides sp. BE1 Belgium Mushroom Yes KY964614 KY964618 Aphelenchoides sp. BE2 Belgium Mushroom Yes KY964615 KY964619 Aphelenchoides sp. E2122 Netherlands Unknown Yes KY964616 KY964620 Aphelenchoides sp. E2133-2 Netherlands Unknown Yes KY964617 KY964621 Aphelenchoides sp. China Pinus thunbergii Yes - KY769087 AspDLDD Aphelenchoides sp. AspNJDH China Pinus thunbergii Yes - KY769088 For scanning electron microscopy (SEM), specimens were fixed with 2% paraformaldehyde (PFA) + 2.5% glutaraldehyde in 0.1M Sorensen buffer. The specimens were dehydrated by passing them through a graded ethanol concentration series of 20, 50, 75, 95, 100% (1 h each), 100% (overnight) and 100% (20 min, next

155 morning). Afterwards, the specimens were critical pointdried with liquid CO2, mounted on stubs with carbon discs and coated with gold (25 nm) before observation with a JSM-840 EM (JEOL, Tokyo, Japan) at 15 kV.

MOLECULAR ANALYSES

DNA samples were prepared according to Li et al. (2008). Three sets of primers (synthesised by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the near full length 18S region and 28S D2-D3 expansion segments of ribosomal RNA genes (rDNA). The 18S region was amplified as two partially overlapping fragments, for the first fragment, 988F (5’-CTC AAA GAT TAA GCC ATG C-3’) and 1912R (5’-TTT ACG GTC AGA ACT AGG G-3’) were used and for the second fragment 1813F (5’-CTG CGT GAG AGG TGA AAT-3’) and 2646R (5’-GCT ACC TTG TTA CGA CTT TT-3’) (Holterman et al., 2006). The 28S D2-D3 region was amplified with the forward primer D2A (5’- ACA AGT ACC GTG AGG GAA AGT TG-3’) and the reverse primer D3B (5’-TCG GAA GGA ACC AGC TAC TA-3’) (De Ley et al., 1999). PCR conditions were as described by Ye et al. (2007) and Li et al. (2008). PCR products were separated on 1.5% agarose gel and visualised by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Invitrogen.

The newly generated sequences of near full length 18S and 28S D2-D3 of rDNA genes were compared with other aphelenchid species available in GenBank (see supplement Table 7-S1) using the BLAST homology search program. The alignment of selected sequences were conducted by MAFFT (Katoh & Standley, 2013) with default parameters and edited by AliView (Larsson, 2014). The best fitted model of DNA evolution and the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were obtained using jModelTest2 (Darriba et al., 2012) under the Akaike Information Criterion (AIC). The models TIM2+I+G and GTR+I+R were selected for the 18S and 28S D2-D3 phylogenetic analyses, respectively.

Bayesian phylogenetic analysis (BI) was carried out using 3.2.3 (Ronquist & Huelsenbeck, 2003). BI analysis for each gene was initiated with a random starting tree and was run with four chains (three heated and one cold) for 2 × 107 generations. After discarding burn-in samples (25%), a 50% majority rule

156 consensus tree was generated visualised using FigTree1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Posterior Probabilities (PP) are given on appropriate clades.

Results

CHARACTERIZATION OF THE HEAD PATTERNS OF THE GENERA IN THE FAMILY

APHELENCHOIDIDAE In this study, the head region characteristics of six populations were studied and visualized (Fig. 7-1.) and data from 72 other species were collected from literature (Table 7-2). The head region of species from different genera in Aphelenchoididae shows a typical hexaradiate cephalic region with six sectors, and the en face view (Fig. 7-2.) is generally very similar to those described by Hooper and Clark (1980). Nonetheless, there are differences among genera and species regarding the annulations of head regions, position of oral apertures, inner labial sensilla, outer labial sensilla, cephalic sensilla and amphids apertures. According to these morphological differences, we classified 78 species from 11 genera into 41 types of head regions, as shown in Table 7-3.

Fig. 7-1. SEM of en face view of six Aphelenchoides populations

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Table 7-2. Overview of molecular and SEM head region information obtained from literature f Aphelenchoididae

No. Species Molecular data Reference 1 Anomyctus xenurus 18S Hooper & Cooke, 1967 2 Aprutides guidettii 18S Pourjam & Bert, 2006 Aphelenchoides besseyi 18S and 28S Yu & Tsay., 2004; Lin et al., 3 2005; Khan et al., 2012 4 A. arachidis - Lesufi et al., 2015 5 A. blastophthorus 18S Hooper & Clark, 1980 6 A. dalianensis - Cheng et al., 2009 7 A. ensete - Swart et al., 2000 8 A. fragariae 18S and 28S Hooper & Clark, 1980 A. fujianensis 18S and 28S Zhuo et al., 2010; Jesus et al., 9 2016 10 A.gorganensis 18S and 28S Miraeiz et al., 2017 11 A. haguei - Swart & Heyns, 1979 A. heidelbergi 18S and 28S Zhao et al., 2006; Carta et al., 12 2016 13 A. nechaleos - Hooper & Ibrahim, 1994 14 A. paradalianensis 18S Cui et al., 2011 15 A. paranechaleos - Hooper & Ibrahim, 1994 16 A. ritzemabosi 18S and 28S Hooper & Clark, 1980 17 A. subtenuis 18S and 28S Deimi et al., 2006 18 A. tuzeti - B’chir 1979 A. variacaudatus 28S Ibrahim & Hooper, 1994; Huang 19 et al., 2012 20 Bursaphelenchus abietinus 18S and 28S Braasch & Schmutzenhofer, 2000 21 B. abruptus 18S and 28S Giblin-Davis et al., 1993 22 B. africanus africanus 18S and 28S Braasch et al., 2007a 23 B. anamurius 28S Akbulut et al., 2007 24 B. antoniae 18S and 28S Penas et al., 2006 25 B. arthuri 18S and 28S Bugermeister et al., 2005 26 B. braaschae 18S and 28S Gu & Wang, 2010 27 B. burgermeisteri 18S and 28S Braasch et al., 2007b 28 B. chengi 18S and 28S Li., PHD thesis 29 B. cocophilus 18S and 28S Giblin-Davis et al., 1989 30 B. curvicaudatus 18S and 28S Wang et al., 2005 31 B. doui 18S and 28S Braasch et al.,2004 32 B. fungivorus 18S and 28S Hooper & Clark, 1980 33 B. gerberae 18S and 28S Giblin-Davis et al., 2006 34 B. glochis - Brzeski & Baujard, 1997 35 B. hellenicus 18S and 28S Skarmoutsos et al., 1998

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36 B. hildegardae 18S and 28S Braasch et al., 2006a 37 B. hofmanni 18S and 28S Braasch et al., 1998 38 B. kevini 18S and 28S Giblin-Davis et al., 1984 39 B. macromucronatus 28S Gu et al., 2008 40 B. obeche 28S Gu et al., 2007 41 B. paracorneolus 18S and 28S Braasch et al., 2000 42 B. paraluxuriosae 18S and 28S Gu et al., 2012 43 B. paraparvispicularis 18S and 28S Gu et al., 2010b 44 B. parapinasteri 18S and 28S Wang & Zhang, 2007 45 B. pinophilus 18S and 28S Brzeski & Baujard, 1997 46 B. platzeri 18S and 28S Giblin-Davis et al., 2006b 47 B. seani 18S and 28S Giblin et al., 1983 48 B. sexdentati 18S and 28S Penas et al., 2004 49 B. sinensis 18S and 28S Palmisano et al., 2004 50 B. singaporensis 18S and 28S Gu et al., 2005 51 B. tusciae 18S and 28S Ambrogioni & Palmisano, 1998 52 B. vallesianus 18S and 28S Braasch et al., 2004 53 B. xylophilus 18S and 28S Nickle et al., 1981 54 B. yongensis 18S and 28S Gu et al., 2006 55 Cryptaphelenchus scolyti - Hooper & Clark, 1980 56 Devibursaphelenchus lini 18S Braasch et al., 2006b Ficophagus aureus 18S and 28S DeCrappeo & Giblin-Davis, 57 2001; Davies et al., 2015 F. laevigatus 18S and 28S DeCrappeo & Giblin-Davis, 58 2001; Davies et al., 2015 59 Laimaphelenchus australis 28S Zhao et al., 2006a 60 L. belgradiensis 18S and 28S Oro, 2015 61 L. cocuccii - Doucet, 1992 62 L. hyrcanus 28S Miraeiz et al., 2015 63 L. persicus 28S Asghari et al., 2012 64 L. preissii 18S and 28S Zhao et al., 2006b 65 Pseudaphelenchus jiaae 18S and 28S Huang et al., 2012 66 P. zhoushanensis 18S and 28S Fang et al., 2016 67 Robustodorus megadorus 18S and 28S Ryss et al., 2013 68 Ruehmaphelenchus asiaticus 28S Braasch et al., 2006c Schistonchus caprifici 18S and 28S DeCrappeo & Giblin-Davis, 69 2001; Davies et al., 2015 S. macrophylla 28S Lloyd & Davies, 1997; 70 DeCrappeo & Giblin-Davis, 2001; Davies et al., 2015 71 Seinura demani 18S Loof & Hooper, 1993 72 S. tenuicauda - Hooper & Clark, 1980

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Fig. 7-2. Basic cephalic plate pattern in Aphelenchoididae after Hooper & Clark 1980

Genus Aphelenchoides

The head region of Aphelenchoides is mostly slightly offset or continuous in some cases. The cuticle of the head region behind the cephalic plate shows annular striations except for A. fujianensis and A. besseyi Ab (see in Table 7-3, A1). The labial disc is surrounded by six equally sized cephalic sectors and show a circular plate comprised of partial fused lateral, subventral and subdorsal cephalic sectors except for A. fujianensis (Zhuo et al., 2010), A. besseyi Ab (Yu & Tsay, 2004) and A. dalianensis (Cheng et al., 2009) which have a distinct separation between each cephalic sector with or without radial grooves emphasizing the individual cephalic sectors.

En face patterns consist of a clearly defined circular oral aperture surrounded by six slightly elevated inner labial sensilla. In most species of Aphelenchoides, there are six Sisyrinchium (a genus of Iridaceae with six oval-round shape petals) petals-like (or a fused hexagram) protuberances surrounding these sensilla, which seem to correspond with the position of the outer labial papillae illustrated by De Grisse (1977) under transmission electron micrographs (TEM). However, the aperture of the outer labial sensilla is invisible. The shape of the outer labial papillae appears to be species specific in some cases, for example, A. gorganensis shows obscure rim-like protuberances (Miraeiz et al., 2017), A. subtenuis has six round-to-trapezoid shape

160 protuberances (Deimi et al., 2006), and A. fragariae has six small round dots (Hooper & Clark, 1980)

The oval-shaped amphidial apertures are similar for all species and positioned in the lateral sectors, just dorsal of the mid line and at the edge of the cephalic plate. Each of the four cephalic sectors bears a prominent cephalic papilla on its outer margin. Each papilla protrudes from a small concavity in the cuticle and a minute central aperture is visible. The most obvious variable characteristic of the labial disc is the extent to which the labial disc is demarcated from the rest of the cephalic plate by a circular groove so that a distinct labial disc is present in all Aphelenchoides species, except, A. ensete (Swart et al., 2000), A. arachidis (Lesufi et al., 2015), A. fragariae (Hooper & Clark, 1980), A. ritzembosi (Hooper & Clark, 1980), A. subtenius (Deimi et al., 2006) and Aphelenchoides sp Asp25 (this document). This groove is associated with by a prominent ring structure only in the species in A. besseyi A030 (this paper) and A. gorganensis (Miraeiz et al., 2017).

Genus Aprutides

Aprutides guidettii is the only species with SEM pictures of the head region in this genus. The head region is high and clearly offset. Head region consists of two parts joined by a strong junction without any annulations, the anterior part resembles to mushroom-like cap with fused six cephalic sectors. The posterior part is wider and thicker than the anterior; it has a truncate cone shape from the middle area towards the anterior end. Four cephalic papilla bearing a small central aperture are located on the two subdorsal and subventral outer margin of the head cap. Pore-like amphid aperture situated at the base of the head cap, in the junction zone of two head parts. En face pattern consisting of oral aperture surrounded by a round- hexangular circle, which may correspond with the position of the inner labial papillae (Pourjam & Bert, 2006). A groove with six slightly slit at each corner of the round- hexangular rim encircles it, which may be associated with outer labial papillae.

Genus Bursaphelenchus

The head region of Bursaphelenchus is often offset by a strong or weak constriction, except for B. cocophilus (Giblin-Davis et al., 1989) and B. platzeri (Giblin-Davis et al., 2006b). The labial annuli can be present or absent. The labial disc is surrounded by cephalic sectors, lateral cephalic sectors are slightly narrower than subdorsal and

161 subventral sectors; they show either a distinct separation from each other or a circular plate comprised of partial fused lateral, subventral and subdorsal cephalic sectors. Radial grooves emphasizing the individual cephalic sectors can be absent or present.

En face patterns consisted of a clearly defined circular oral aperture surrounded by six slightly elevated inner labial sensilla. Encircling them, there are six round or oval- rectangle protuberances where the aperture of outer labial sensilla are visible. The protuberances fused to a ring in some species. Pore-like amphidial apertures, dorso-medially located on lateral cephalic sectors and a slightly-elevated cephalic papilla clearly resolved on the outer margin of each subdorsal and subventral cephalic sector. However, the labial disc of B. abruptus, B. cocophilus (Giblin-Davis et al., 1989), B. platzeri (Giblin-Davis et al., 2006b) and B. kevini (Giblin-Davis et al., 1984) are different from the above description. In B. abruptus, a circular and sunken depression surrounds the oral aperture, labial sensilla are often obscured. According to the original description (Giblin-Davis et al., 1993), analysis of several SEM en face patterns indicates a full complement of six inner labial papillae adjacent to the oral aperture and six outer labial papillae just inside the sunken depression. B. cocophilus presents an oral aperture surrounded by a labial disc that is specialized by dorso-ventral elongation and small indentations on each lateral side (Giblin-Davis et al., 1989). Inner labial sensilla were not resolved on the labial disc. B. platzeri’s en face pattern (visible under SEM) is composed of a clearly defined circular oral aperture surrounded by a dorsoventrally elongated labial disc, with a long indentation on each lateral side and a small indentation on each dorsal and ventral side. The inner labial sensilla are not resolved neither on nor at the periphery of the labial disc but may be integrated somehow with indentations (Giblin-Davis et al., 2006b). Outer labial sensilla obscured; en face patterns (under SEM) suggests two lateral outer labial sensilla open mediolaterally onto a circular lip sector disc. B. kevini’s en face patterns consist of a clearly defined circular oral aperture surrounded by six slightly elevated inner labial sensilla. Six outer labial papillae appear as round dots, surrounded by a ring-shaped lip sector disc (Giblin-Davis et al., 1984).

Genus Cryptaphelenchus

The head region of Cryptaphelenchus is offset by a constriction. The slightly trapezoid cuticle of the head region, behind the cephalic plate, shows clear annulations (Hooper & Clark, 1980). The labial disc is surrounded by six cephalic

162 sectors, which are equal in size and show a circular plate comprising fused lateral, subventral and subdorsal cephalic sectors. Cryptaphelenchus presents pore-like amphidial apertures that are dorso-medially located on lateral cephalic sectors; a slightly elevated cephalic papilla clearly resolved on the outer margin of each subdorsal and subventral cephalic sector is also present. En face pattern consists of a dorso-ventral slit. Other characters of labial section are not resolved.

Genus Devibursaphelenchus

Devibursaphelenchus lini (Braasch et al., 2006b) is the only species in this genus with SEM pictures. The slightly trapezoid cuticle of the head region behind the cephalic plate shows transverse striae. The labial disc is surrounded by six cephalic sectors, i.e., two lateral, two subdorsal and two subventral sectors out of which the lateral sectors are narrower than the subdorsal and subventral. Pore-like amphidial apertures are located on lateral cephalic sectors, just dorsal to the mid line and at the edge of the cephalic plate. Each of the other four cephalic sectors bears a slightly elevated cephalic papilla on its outer margin. En face patterns consist of an oral aperture surrounded by a hexaradiate labial disc with six radial depressions and demarcated by a sunken depression. The labial disc shows six inner labial sensilla adjacent to the stoma. Outer labial sensilla were not resolved on the labial disc.

Genus Ficophagus

Within the genus Ficophagus, only F. aureus and F. laevigatus (DeCrappeo & Giblin-Davis, 2001) have SEM pictures with continuous head regions. The cuticle of the head region behind the cephalic plate shows clear annulations. The labial disc is surrounded by fused cephalic sectors i.e., two subdorsal, two lateral, two subventral. Amphidial apertures are dorso-medially located on each lateral cephalic sector; each of the other cephalic sectors i.e., two subdorsal and two subventral cephalic sectors, bears a prominent cephalic papilla on its outer margin. Each papilla protrudes from a small concavity in the cuticle and in some species a minute central aperture is visible. En face pattern consists of a labial disc with clearly defined oral aperture surrounded by six slightly elevated inner labial sensilla. Six obscured round-dots like outer labial sensilla are located on the fused cephalic sectors surrounding the labial disc. However, the apertures of the outer labial sensilla are not resolved (DeCrappeo & Giblin-Davis, 2001, Davies et al., 2015).

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Genus Laimaphelenchus

The head region of Laimaphelenchus is rounded to hemispherical, often continuous or slightly offset. The cuticle of the head region behind the cephalic plate shows clear annulations. The labial disc is indistinct, fused with lips except for L. helicosoma a distinct labial disc is present because the lip sector disc is demarcated from the rest of the cephalic plate by a circular groove (Hooper & Clark, 1980). . The six cephalic sectors are equal in width, amalgamated and marked by double, well-developed ridges (showed as solid line in Table 7-3. L5.) or distinct grooves (showed as dotted line in Table 7-3. L1 to L4.) except for L. preissii. En face patterns consist of a clearly defined circular oral aperture surrounded by six slightly elevated inner labial sensilla, outer labial sensilla are invisible. The oval-shaped amphidial apertures are in the lateral sectors, just dorsal of the mid line but are only visible in L. preissii, L. hyrcanus, L. australis and L. helicosoma. The cephalic papilla are absent except for the latter species and some individuals of L. preissii where the cephalic papilla are at the edge of the cephalic plate.

Genus Pseudaphelenchus

The head region of Pseudaphelenchus is rounded and continuous but appears to be slightly offset in light microscopy. The cuticle of the head region behind the cephalic plate shows no annulations. The labial disc is surrounded by six cephalic sectors which are equal in size and show a circular plate comprised of partial fused lateral, subventral and subdorsal cephalic sectors. The oval-shaped amphidial apertures are positioned dorsolaterally on the edges of the cephalic plate of the lateral cephalic sectors. Each of the other four cephalic sectors bears a prominent cephalic papilla on its outer margin. En face patterns consist of a clearly defined circular oral aperture surrounded by six slightly elevated inner labial sensilla. The elevation encircling them, may correspond with the position of the outer labial papillae.

Genus Robustodorus

Robustodorus is a monotypic genus (R. megadorus). The head region of R. megadorus is hemispherical and set-off by a deep constriction without annulations. The labial disc is indistinct, surrounded by six cephalic sectors, which are equal in size and show a smoothly circular plate comprised of fused lateral, subventral and subdorsal cephalic sectors with obscure grooves. En face pattern consists of a clearly defined circular

164 oral aperture surrounded by six inner labial sensillar pores which are encircled by a slightly hexagram shaped protuberance where may correspond with the position of outer labial sensilla. Pore-like amphids are small and located in the lateral sectors, just dorsal of the mid line and at the edge of the cephalic plate. The cephalic papilla are clearly resolved on each subdorsal and subventral cephalic sector and form the outline of a square (Ryss et al., 2013).

Genus Ruehmaphelenchus

The head region of Ruehmaphelenchus is often offset by a constriction. The cephalic annuli are not present in the head region. The labial disc is surrounded by six cephalic sectors which are almost equal in size and shows a distinct separation from each other, central ribs emphasis the individual cephalic sectors. Amphidial apertures and cephalic papilla are not visible under SEM. En face patterns consist of a clearly defined circular oral aperture surrounded by six elevated inner labial sensilla, encircled by six round or trapezoid-square protuberances fused into a ring. The apertures of outer labial sensilla are invisible. Labial disc is surrounded by an almost circular plate, an evident lip sector disc (Braasch et al., 2006c).

Genus Schistonchus

The head region of Schistonchus is slightly offset and without cephalic annulations. The labial disc is distinctly ringed and surrounded by fused cephalic sectors (two subdorsal, two lateral, two subventral). Two dorso-lateral amphidial apertures obscured as covered by possible secretions, a slightly elevated cephalic papilla on the outer margin of each subdorsal and subventral cephalic sector. The labial disc with clearly defined oral aperture surrounded by six slightly elevated inner labial sensilla and a distinct labial disc without visible outer labial sensilla (Lloyd & Davies, 1997; De Crappeo & Giblin-Davis, 2001; Davies et al., 2015).

Genus Seinura

The head region of Seinura is offset by a constriction. The slightly trapezoid cuticle of the head region behind the cephalic plate shows clear annulation. The labial disc is surrounded by six cephalic sectors (two lateral, two subdorsal and two subventral) where the lateral sectors are narrower than the subdorsal and subventral sectors, with radial grooves emphasizing the individual cephalic sectors. Seinura has pore-like amphidial apertures, dorso-medially located on lateral cephalic sectors. A slightly

165 elevated cephalic papilla is also visible, clearly resolved on the outer margin of each subdorsal and subventral cephalic sector. En face pattern consists of stomatal aperture surrounded by six inner liplets where the aperture of inner labial sensilla are vaguely visible in S. demani (Loof & Hooper, 1993). In S. tenuicaudata, there are lobes between the stomatal aperture and the lateral liplets (Hooper & Clark 1980).

MOLECULAR PROFILES AND PHYLOGENETIC STATUS

Respectively, 30 and 25 sequences of 18S and 28S markers were generated. Together with 237 18S and 307 28S sequences from the GenBank, 404 taxa in total were included in the phylogenetic concatenated alignment, resulting in 1982 characters for 18S and 1151 characters for 28S D2-D3. The BI tree (Fig. 7-3.) contains five major clades with high posterior probabilities. Thhe topology is similar to Kanzaki & Giblin-Davies (2012) and their clade names are used in this study. Pseudaphelenchus (Clade 1) is sister to all other taxa with maximal support. The clade 2 (PP: 100) consists of Robustodorus, Aprutides, all Ficophagus, Schistonchus, Martininema, Laimaphelenchus, and all Aphelenchoides species, except for A. stammeri. This clade can be divided into two subclades, 2a and 2b, also maximally suported. The Clade 2a comprises Ficophagus, Martininema and most Aphelenchoides species, with Robustodorus megadorus and A. subtenuis as sister taxa at an early-branching position. The Clade 2b contains all Schistonchus and Laimaphelenchus species as well as several Aphelenchoides species. Clade 3 (PP: 89) comprises the predator genera Ektaphelenchus, Ektaphelenchoides, Devibursaphelenchus, Seinura and Anomyctus, insect parasites genera Noctuidonema and Peraphelenchus, the fungivorous genus Cryptaphelenchus, A. stammeri and Bursaphelenchus abruptus. Clade 4 (PP: 100) is composed by all Bursaphelenchus spp., except for B. abruptus, and Sheraphelenchus is also imbedded in this clade. Finally, clade 5 (PP: 100), sister to clade 4, contains all Ruehmaphelenchus species.

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1 Table 7-3. Different type of head region in genera of Aphelenchoididae.(Present = P, Invisible = I) Outer Cephalic Annul Amphidial Cephalic Grooves Labial disc labial Type En face and lateral view Species/Population ation apertures papillae or ridges sectors sensillae

Aphelenchoides besseyi Clearly Slightly Clear Not No P P Groove defined A1 Ab offset separated resolved

A. fujianensis

Clearly Slightly Clear Not Yes P P Groove defined A2 A. dalianensis offset separated resolved

Faint Clearly Slightly Sisyrinchi Fused Yes P P No defined A3 A. subtenius offset um-petals

Clearly Continuo Partial Six round Yes P P No defined A4 A. fragariae us fused dots

Aphelenchoides sp. Asp25 Clearly A. ensete, Slightly Partial Yes P P No defined Hexagram A5 offset fused A. arachidis, A. ritzembosi,

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Clearly Aphelenchoides sp. AspX, defined Sisyrinchi Slightly Partial Yes P P No with um-petals A6 Aphelenchoides sp. 12Asp offset fused groove like A. heidelbergi

Clearly Sisyrinchi A. besseyi A030 Slightly Partial Yes P P No defined um-petals A7 offset fused A. besseyi with ring like

Clearly A. gorganensis Slightly Partial Not Yes P P No defined A8 offset fused resolved with ring

Clearly defined A. besseyi A003 Slightly Partial Yes P P No with Hexagram A9 offset fused A. haguei groove

Clearly defined Slightly Partial Not Yes P P No with A10 A. blastophthorus offset fused resolved groove

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Clearly defined Slightly Partial Yes P P Groove with Hexagram A11 Aphelenchoides sp. 18Asp offset fused groove

Aphelenchoides sp. 14Asp A. xui, A. nechaleos, Clearly defined Sisyrinchi A. paradanlianensis, Slightly Partial Yes P P Groove with um-petals A12 offset fused A. paranechaleos, groove like

A. tuzeti, A. varicaudatus, A. stellatus,

Strongly Not Fused No P P Groove Indistinct Ro1 Robustodorus megadorus Offset resolved

Laimaphelenchus Continuo Not Fused No P I Ridges Indistinct L1 us resolved australis

Continuo Not Fused No I I Ridges Indistinct L2 L. coucucci us resolved

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Clearly defined Continuo Not Fused No P P Ridges with L3 L. helicosoma us resolved groove

Continuo Not Fused Yes P P Ridges Indistinct L4 L. belgradiensis us resolved

Slightly Not Fused Yes P I Groove Indistinct L5 L. hyrcanus offset resolved

Slightly Not Fused Yes P P No Indistinct L6 L. preissii offset resolved

Ficophagus aureus Continuo Partial Clearly Six round Yes P P No F1 us fused defined dots F. laevigatus

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Clearly Schistonchus caprifici Continuo Not Fused No P P No defined Sc1 us resolved S. macrophylla with a ring

Clearly Continuo Partial defined Not Yes P P Ridges B1 Bursaphelenchus platzeri us fused with resolved groove

Continuo Clearly Not Fused Yes P P No B2 Unavailable B. cocophilus us defined resolved

B. abietinus, B. arthuri, Six oval- B. paracorneolus, Clear Clearly rectangle offset No P P Groove B3 separated defined protubera B. sinensis, nces B. hellenicus, B. yongensis

B. anamurias, Six oval- Clear Clearly rectangle offset No P P Groove B4 B. hofmani, separated defined protubera nces B. parapinasteri

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Six oval- B. africanus Clear Clearly rectangle offset Yes P P No B5 separated defined protubera B. burgermeisteri nces

Clearly defined Not offset Fused No P P No B6 B. abruptus with resolved depression

B. antoniae, Six oval- Partial Clearly rectangle offset No Present P No B7 B. chengi, fused defined protubera nces B. pinophilus

Six oval- B. vallensianus, Partial Clearly rectangle B8 offset No P P Groove B. sexdentati fused defined protubera nces

B. corneolus corneolus,

B. glochis, Six oval- B. paraluxuriose, Partial Clearly rectangle B9 offset Yes P P No B. paraparvispicularis, fused defined protubera nces B. singaporensis, B. xylophilus

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Partial Six round offset Yes P P No Indistinct B10 B. seani fused dots

B. braaschae,

B. doui, Six oval- B. gerberae, Partial Clearly rectangle B11 offset Yes P P Groove B. macromucronatus, fused defined protubera nces B. tusciae, B. fungivorus

Partial Clearly Six round offset Yes P P Groove B12 B. obeche fused defined dots

Clearly Partial Six round offset Yes P P Groove defined B13 B. kevini fused dots with ring

Six oval- Clearly Ruehmaphelenchus Clear rectangle Offset No P P Groove defined Ru1 separated protubera with ring asiaticus nces

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Slightly Partial Clearly Yes P P No Six lobes Se1 Seinura tenuicauda Offset fused defined

Partial Clearly Offset Yes P P Groove Six lobes Se2 S. demani fused defined

Partial Clearly Offset Yes P P No Six lobes D1 Devibursaphelenchus lini fused defined

Strong Clearly Not Fused No P P No Ap1 Aprutides guidettii offset* defined resolved

Pseudaphelenchus Slightl Not P Partial Clearly y No P P No resolve zhoushanensis fused defined 1 Offset d P. jiaae

2 3

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Fig.7-3. Mapping of the head patterns types on the concatenated phylogenetic tree of Aphelenchoididae

PHYLOGENETIC STATUS AND HEAD REGION CHARACTERS

In order to reveal if the head patterns agree with molecularly defined clades, information obtained by available detailed SEM pictures (see Fig. 7-3.) has been mapped on the phylogenetic tree. The Clade 1 only contains Pseudaphelenchus, with a head region with smooth and rounded head with fused cephalic sectors. En face patterns consist of a clearly defined circular oral aperture surrounded by six slightly elevated inner labial sensilla with a circle encircling which may correspond with the position of the outer labial papillae.

Clade 2 (see Fig. 7-4.) shows the paraphyly of Aphelenchoides, as Ficophagus,

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Laimaphelenchus, Martininema, Schistonchus, Robustodorus and Aprutides are embedded within Aphelenchoides sequences. Schistonchus sensu stricto is placed in Clade 2b while Ficophagus, Martininema, Robustodorus and Aprutides are positioned within Clade 2a. The different patterns of the head region were tabulated in 12 types for the genus Aphelenchoides, 6 types in Clade 2a and 6 types in Clade 2b. The type A11 is the most typical Aphelenchoides pattern, where not only the labial disc is clearly defined by a groove but also the outer labial is Sisyrinchium-petals like. These 11 types of head regions relatively similar to the general structures described by as Hooper and Clark (1980). Clearly, head patterns are not corresponding to molecularly defined clades and several patterns have evolved independently, for example , the type A5 Aphelenchoides can be found in both clades 2a and 2b. Remarkably, two different head patterns have been found for A. besseyi has been reported as a parasite of rice (Oryza sativa L.), strawberry (Fragaria grandiflora Ehrn.), bird’s-nest fern (Asplenium nidus L.), and many other ornamental plants (Wang et al., 1993, Yu & Tsay, 2004, Tsay et al., 1995, Sánchez-Monge et al., 2015). This variation agrees either with the great intraspecific heterogeneity as known for this species or is an indication that A. besseyi is a species complex. Remarkable intraspecific heterogeneity includes host range (Sturhan, 1971) and mode of reproduction of A. besseyi, i.e. ie parthenogenesis (Bridge et al., 1990), amphimictic (Franklin & Siddiqi 1972: Huang et al., 1979; Wang et al., 1993; Lin et al., 1992) or both parthenogenesis and amphimixis Tzeng (2005) (Tzeng & Lin 2005). The most recent publication on A. besseyi’s reproductive behaviour also highlights that this species has more than one reproductive strategy, moreover, two reproductive modes in a single host race were documented for the first time (Hsieh et al., 2012). These phenomena indicate that A. besseyi is probably a species complex, as illustrated by Jesus et al (2016) that two groups were originally thought to be A. besseyi, but one of them, i.e., Group-forage, was later on identified as A. fujianenesis.

Within the A. besseyi populations (i.e., group-Rice) (see Table 7-4.), DJ11 was is isolated from Phaseolus vulgaris from Costa Rica and shows different morphometrics, with a larger PUS/VA ratio than the other populations in this group (Jesus et al., 2016). In this study, the DJ11 population clustered with five A. besseyi sequences (also isolated from Phaseolus vulgaris in Costa Rica) and four A. besseyi populations isolated from Asplenium nidus in Taiwan. This clade appears with a 100% posterior probability within the group-Rice clade (Fig. 7-5.), other sequences isolated from rice in Costa Rica clustered with other populations within this clade. Some populations of the so called Group-forage, considered as A.

176 fujianensis, clustered with the original sequence of A. fujianensis.

The SEM pictures of Aphelenchoides besseyi A030 (from rice) and Aphelenchoides besseyi A003 (from beans) show an overall alike morphology, except for the head region. A. besseyi A030 is closer to A7 type pattern with the labial disc clearly defined by a prominent ring surrounding the Sisyrinchium-petals like outer labial sensilla, while the labial disc of A. besseyi A003 was clearly defined by a typical groove surrounding the hexagram shape outer labial sensilla resembling the A9 pattern. Based only on this condition, these two populations can be easily distinguished.

Table 7-4. Origin and host of different populations of Aphelenchoides besseyi Reference code Country of origin Substrate/host Genbank accession numbers 18S 28S Aphelenchoides besseyi “Beans” A. besseyi A003 Costa Rica Phaseolus vulgaris KX356697 KX356753 A. besseyi A011 Costa Rica Phaseolus vulgaris KX356698 KX356755 A. besseyi A016 Costa Rica Phaseolus vulgaris KX356699 KX356756 A. besseyi A042 Costa Rica Phaseolus vulgaris KX356702 - A. besseyi A068 Costa Rica Phaseolus vulgaris KX356703 KX356765 A. besseyi DJ11 Costa Rica Phaseolus vulgaris - KT692694 A. besseyi Am Taiwan Asplenium nidus - HQ540538 A. besseyi Fgk Taiwan Asplenium nidus KT943535 - A. besseyi Fm Taiwan Asplenium nidus KT454962 - A. besseyi Fsx Taiwan Asplenium nidus KT943534 - Aphelenchoides besseyi “Rice” A. besseyi A004 Costa Rica Oryza sativa - KX356754 A. besseyi A028 Costa Rica Oryza sativa - KX356758 A. besseyi A029 Costa Rica Oryza sativa - KX356759 A. besseyi A030 Costa Rica Oryza sativa KX356700 KX356760 A. besseyi A031 Costa Rica Oryza sativa KX356701 KX356761 A. besseyi A033 Costa Rica Oryza sativa - KX356762 A. besseyi A040 Costa Rica Oryza sativa - KX356763 A. besseyi A041 Costa Rica Oryza sativa - KX356764 A. besseyi A081 Costa Rica Oryza sativa - KX356766 A. besseyi A106 Costa Rica Oryza sativa - KX356767 A. besseyi A107 Costa Rica Oryza sativa - KX356768 A. besseyi A118 Costa Rica Oryza sativa - KX356769 A. besseyi A121 Costa Rica Oryza sativa - KX356770 A. besseyi A124 Costa Rica Oryza sativa - KX356771

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A. besseyi A125 Costa Rica Oryza sativa - KX356772 A. besseyi A127 Costa Rica Oryza sativa KX356704 KX356773 A. besseyi 754a_3fe Russia Unknown - DQ328684 A. besseyi A_bes_CH China Oryza sativa KX356705 KX356774 A. besseyi A_bes_IT Italy Unknown KX356706 KX356775 A. besseyi A_bes_Tk Turkey Unknown KX356707 KX356776 A. besseyi Ae Taiwan Oryza sativa - HQ540535 A. besseyi Al Taiwan Oryza sativa - HQ540537 A. besseyi Ap Taiwan Oryza sativa - HQ540539 A. besseyi Rdg Taiwan Oryza sativa KT943536 - A. besseyi RI Taiwan Oryza sativa KT454963 - A. besseyi DJ06S Japan Oryza sativa KT692669 KT692689 A. besseyi DJ07S Japan Oryza sativa KT692671 KT692690 A. besseyi DJ12S Brazil Oryza sativa KT692675 KT692696 A. besseyi DJ15S Brazil Oryza sativa KT692679 KT692700 A. besseyi DJ18S Spain Oryza sativa KT692682 KT692703 A. besseyi China Setaria italica - KP757368 A. besseyi S1 Brazil Brachiaria - KT692704 brizantha A. besseyi S12 Brazil Brachiaria - KT692708 ruziziensis A. besseyi S2 Brazil Brachiaria - KT692705 humidicola Aphelenchoides sp. Be Unknown Unknown GU337995 - Aphelenchoides sp. TG102006 Vietnam Polianthes tuberosa - EU084037

The head region of A. besseyi Ab (Table 7-3. A1) shows a distinct separation from each cephalic sector, differing from the head region of A. fujianensis which shows partial fused cephalic sectors, however, the SEM pictures of the original description are not clear. Despite the contrasting in reproduction strategies, host ranges and the geographic distributions, the combination of molecular data and morphological characters is efficient to diagnose A. besseyi and A. fujianensis, especially the pattern of the head region.

The monotypic genus Robustodorus is characterized by the clearly offset cephalic region and an exceptionally robust stylet with massive, rounded basal knobs (Ryss et al., 2013). Surprisingly, it forms a strongly supported clade (PP: 100) sister to A. subtenius which has slender stylet with swelling and typical Aphelenchoides head pattern similar to A. fragariae (Hooper and Clark, 1980) and A. ritzemabosi (Deimi et al., 2006). Regardless the great similarity with other Aphelenchoides species, the outer labial sensilla of A. subtenuis are not similar to those in A. fragariae or A. ritzemabosi, and because of its faint Sisyrinchium-petals

178 like shape, another type, i.e., A3 type was assigned to A. subtenuis in this study. The head region of Robustodorus, which is clearly resolved in the redescription by Ryss et al (2013), clearly differs from A. subtenuis by the deep constriction without annulations, obscure grooves on cephalic sectors and the faint hexagram prominent of the outer labial sensilla.

The genus Aprutides is also clustered into Clade 2a, and its phylogenetic relationships remains unresolved in the concatenated tree. Aprutides is characterized by the lightly sclerotized stylet with sclerotized attachment points for the protractor muscles (Hunt, 1993). After comparing to other genera in Aphelenchoididae, Aprutides has a unique head region structure, which consists of two divided parts and a junction zone where the amphid apertures are located.

The genus Schistonchus was recently divided into three distinct genera: Schistonchus (sensu stricto), Ficophagus and Martininema based on a combined analysis of molecular and morphological data (Davies et al., 2015); Ficophagus and Martininema are nested in a robust Aphelenchoides clade 2a (PP:100) and such placement of Ficophagus is further supported by its head pattern similar to A. fragariae, Martininema’s head morphology is unknown. Schistonchus (sensu stricto) is monophyletic (PP: 100), sister to Laimaphelenchus in a moderately supported clade (PP: 95) in Clade 2b. The comparison of the en face patterns of both genera showed that the labial disc has invisible outer labial sensilla and indistinct cephalic sectors.

The Clade 3 (see Fig. 7-3.) comprises more complicated paraphyletic relationships regarding the predator genera Ektaphelenchus, Ektaphelenchoides, Devibursaphelenchus, Seinura and Anomyctus, the insect parasitic genera Noctuidonema and Peraphelenchus, and genus Cryptaphelenchus, Aphelenchoides stammeri and Bursaphelenchus abruptus, the latter three being fungivorous taxa. Devibursaphelenchus have en face patterns (Hooper & Clark 1980) that consist of the stomatal aperture surrounded by six inner liplets, similar to the head patterns that found on Seinura and Anomyctus (Hooper & Cooke 1967) which are predators.

Cryptaphelenchus on the other hand, is clustered into a monophyletic clade in Clade 3, and although the SEM of its head region seems convoluted, this may be partly distorted (Hooper & Clark 1980). The split-like oral aperture is different from other fungivorous genera such as Aphelenchoides and Bursaphelenchus, where the oral aperture is rounded. Bursaphelenchus abruptus has a circular and sunken depression surrounding the oral aperture, which is different from all other Bursaphelenchus species. However, the head region of 179

Aphelenchoides stammeri is not available; hence, the comparison of these two special members in this clade is not possible.

Fig. 7-4. Phylogenetic relationships of Aphelenchoididae from a concatenated file of 18S and D2-D3 region of 28S. The taxa in clade 2a and 2b are shown and the number of sequences is given. Other clades were collapsed in triangular. Aphelenchus avenae and Paraphelenchus acontioides served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

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Fig. 7-5. Phylogenetic relationships of Aphelenchoididae from a concatenated file of 18S and D2-D3 region of 28S. The populations of Aphelenchoides besseyi in Clade 2b are shown. Posterior probability values exceeding 50% are given on appropriate clades.

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Fig. 7-6. Phylogenetic relationships of Aphelenchoididae from a concatenated file of 18S and D2-D3 region of 28S. The taxa in clade 4 are presented together with the head region pattern f available. Other clades were collapsed (triangular-. Aphelenchus avenae and Paraphelenchus acontioides served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

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Discussion

The cephalic region of Aphelenchoididae is typically hexaradiate with cephalic sectors of almost equal size. The cephalic sclerotization is usually weak, while the oral aperture is circular to oval in shape and is surrounded by a circle of six inner labial papillae (Hooper & Clark, 1980). There are usually six bumps external to the oral disc that may represent the outer labial papillae and the four cephalic papillae are in submedian position. The amphidial apertures are small and pore-like and are located dorsosublaterally on the lateral cephalic sectors (Hooper & Clark, 1980). However, a comprehensive comparison based on multiple species and genera of Aphelenchoididae revealed considerable differences within and between genera and species.

Cephalic sectors

The cephalic sectors of most Aphelenchoididae genera are almost equal in size and partially fused to form a typical hexagram shape, however, in one undescribed species of Entaphelenchiae only four head sectors were seen (Giblin & Kaya 1983). In Devibusaphelenchus, Ektaphelenchoides and Ektaphelenchus, the lateral cephalic sectors are clearly narrower than the subventral and subdorsol cephalic sectors which makes the view of head region wider and flatter under light microscopy. The cephalic sectors of some Bursaphelenchus and Aphelenchoides species are clearly separated from each other, which may be useful for species identification. In some cases the cephalic sectors are not visible.

The presence of ridges or grooves on the cephalic sectors is put forward as an important diagnostic character in Laimaphelenchus (Ryss et al., 2013). A three-dimensional reconstruction based on serial TEM sections of tissues and cuticle of the stomatostylet apparatus of Aphelenchus avenae (Ragsdale et al., 2008) showed that the ridges or grooves may correspond to the gap in the epidermal syncytia HypC.

Oral aperture

The oral aperture is from circular to oval shape in the centre of the labial disc. However, in the predator genera Anomyctus, Devibusaphelenchus and Seinura, the en face view of the oral opening is a hexaradiate stoma with surrounding liplets that can only be observed in other predator genera of Aphelenchoididae (see above). Hence, such arrangement is probably associated with their predatory behaviour (Hooper & Clark 1980). Furthermore, this feature is also common in some predator genera of Dorylaimida such as Amblydorylaimus isokaryon

183

(Elshishka et al., 2015), Eudorylaimus sp. (Jairajpuri & Ahmad, 1992) and Nygolaimina (Shafqat et al., 1994) in which this structure may be beneficial to stretch out the long and robust odontostyle.

Inner labial sensilla

The inner labial sensilla of Aphelenchoididae have small slit-like openings around the prestomal opening. The openings of the inner labial sensilla in Ficophagus are bigger than in any other Aphelenchoididae and are invisible in several species of Bursaphelenchus, Devibusaphelenchus and Seinura.

Outer labial sensilla

Distally, the outer labial sensilla generally end blindly in the head cuticle (De Grisse et al., 1979, Ragsdale et al., 2009) and the position of the outer labial papillae seem to correspond protuberances encircling the inner labial sensilla, which can be with of different shape(Hooper & Clark 1980). Within Aphelenchoididae, the apertures of the outer labial papillae seem to be only visible in most Bursaphelenchus species.

Cephalic sensilla

Two different types of receptors are present in the cephalic sensilla of Aphelenchoides (De Grisse et al., 1979). The main branch of the sensillar canal follows the head contour and ends blindly in the head cuticle, and shortly behind the tip of the outer labial sensillum, a narrow side branch splits off, leading to a pore on top of a small papilla where it is located at the outer margin of each subdorsal and subventral cephalic sector (Coomans & De Grisse 1981). However, these small papilla are invisible in some Laimaphelenchus species, which may indicate that the narrow side branch also ends blindly.

Amphids apertures

The amphidial apertures of Aphelenchoididae are small and pore-like, situated dorsally from the median axis of the lateral line (Hooper & Clark 1980). These apertures are obscure in some Laimaphelenchus species, while in some species of Bursaphelenchus (e.g. Bursaphelenchus kevini) the amphidial opening tends to be closer to the oral aperture, despite this position, they are not as close as in various Tylenchoidea (Sher & Bell, 1975; Stone, 1975; De Grisse 1977; De Grisse 1979).

In general, the head region of Aphelenchoididae, especially in Bursaphelenchus and

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Aphelenchoides, looks quite similar in light microscopy. In this study, the comparison of the head region revealed that they are more diverse than previously thought. For Bursaphelenchus, the main difference is the absence of the aperture of the outer labial sensilla. Species within Aphelenchoides show low inter-specific and high intra-specific morphological variability, causing a considerable overlapping in morphology and morphometrics (Hockland, 2001) which has led to several misidentifications, taxonomic confusions and the presence of cryptic species (Kanzaki & Giblin-Davis, 2012). Some attempts have shown that combination of SEM and other datasets can improve identification accuracy: e.g. Aphelenchoides fujianensis was recognized from “Aphelenchoides besseyi” complex (Jesus et al., 2016).

Despite this comprehensive overview, the availability of SEM and molecular data of Aphelenchoididae is still limited, for example for Entaphelenchinae detailed SEM pictures and molecular data are still completely absent..Several authors (see DeCrappeo & Giblin-Davis, 2001; Davies, 2010), suggested that the cephalic plate morphology could be informative at genus level. However, as shown in this study, the ultrastructure of the head region does not agrees with genus delimitation but it can reflect particular species-level differences.

To have a further understanding of the various structures, TEM in combination with 3D is warranted (e. g. the cuticular groove around labial papillae of Aphelenchoides, the ridges on the cephalic sectors of Laimaphelenchus and the ring). Insights related to the head region at a cellular level may also shed light on the understudied ecological relationships among the highly diverse Aphelenchoididae.

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Table 7-S1. Species/Populations used in concatenated tree

Accession number Subfamily References code 18S 28S Anomyctus xenurus FJ040413 - Aphelenchoidinae Aphelenchoides besseyi 754a_3fe - DQ328684 Aphelenchoidinae Aphelenchoides besseyi 98 AY508035 AY508109 Aphelenchoidinae Aphelenchoides besseyi 99 - EU325682 Aphelenchoidinae Aphelenchoides besseyi A_bes_CH KX356705 KX356774 Aphelenchoidinae Aphelenchoides besseyi A_bes_IT KX356706 KX356775 Aphelenchoidinae Aphelenchoides besseyi A_bes_Tk KX356707 KX356776 Aphelenchoidinae Aphelenchoides besseyi A003 KX356697 KX356753 Aphelenchoidinae Aphelenchoides besseyi A004 - KX356754 Aphelenchoidinae Aphelenchoides besseyi A011 KX356698 KX356755 Aphelenchoidinae Aphelenchoides besseyi A016 KX356699 KX356756 Aphelenchoidinae Aphelenchoides besseyi A028 - KX356758 Aphelenchoidinae Aphelenchoides besseyi A029 - KX356759 Aphelenchoidinae Aphelenchoides besseyi A030 KX356700 KX356760 Aphelenchoidinae Aphelenchoides besseyi A031 KX356701 KX356761 Aphelenchoidinae Aphelenchoides besseyi A033 - KX356762 Aphelenchoidinae Aphelenchoides besseyi A040 - KX356763 Aphelenchoidinae Aphelenchoides besseyi A041 - KX356764 Aphelenchoidinae Aphelenchoides besseyi A042 KX356702 - Aphelenchoidinae Aphelenchoides besseyi A068 KX356703 KX356765 Aphelenchoidinae Aphelenchoides besseyi A081 - KX356766 Aphelenchoidinae Aphelenchoides besseyi A106 - KX356767 Aphelenchoidinae Aphelenchoides besseyi A107 - KX356768 Aphelenchoidinae Aphelenchoides besseyi A118 - KX356769 Aphelenchoidinae Aphelenchoides besseyi A121 - KX356770 Aphelenchoidinae Aphelenchoides besseyi A124 - KX356771 Aphelenchoidinae Aphelenchoides besseyi A125 - KX356772 Aphelenchoidinae Aphelenchoides besseyi A127 KX356704 KX356773 Aphelenchoidinae Aphelenchoides besseyi Ab - HQ540534 Aphelenchoidinae Aphelenchoides besseyi AChoBes1 JQ957878 - Aphelenchoidinae Aphelenchoides besseyi AChoBes2 JQ957877 - Aphelenchoidinae Aphelenchoides besseyi Ae - HQ540535 Aphelenchoidinae Aphelenchoides besseyi Af - HQ540536 Aphelenchoidinae Aphelenchoides besseyi Al - HQ540537 Aphelenchoidinae Aphelenchoides besseyi Am - HQ540538 Aphelenchoidinae Aphelenchoides besseyi Ap - HQ540539 Aphelenchoidinae Aphelenchoides besseyi As - HQ540540 Aphelenchoidinae Aphelenchoides besseyi Au - HQ540541 Aphelenchoidinae Aphelenchoides besseyi DJ06S KT692669 KT692689 Aphelenchoidinae Aphelenchoides besseyi DJ07S KT692671 KT692690 Aphelenchoidinae Aphelenchoides besseyi DJ11 - KT692694 Aphelenchoidinae Aphelenchoides besseyi DJ12S KT692675 KT692696 Aphelenchoidinae Aphelenchoides besseyi DJ15S KT692679 KT692700 Aphelenchoidinae Aphelenchoides besseyi DJ18S KT692682 KT692703 Aphelenchoidinae 186

Table 7-S1 (continued) Aphelenchoides besseyi Fgk KT943535 - Aphelenchoidinae Aphelenchoides besseyi Fm KT454962 - Aphelenchoidinae Aphelenchoides besseyi Fsx KT943534 - Aphelenchoidinae Aphelenchoides besseyi HB clone 1 - KP757368 Aphelenchoidinae Aphelenchoides besseyi HB clone 2 - KP757369 Aphelenchoidinae Aphelenchoides besseyi HB clone 3 - KP757370 Aphelenchoidinae Aphelenchoides besseyi Rdg KT943536 - Aphelenchoidinae Aphelenchoides besseyi RI KT454963 - Aphelenchoidinae Aphelenchoides besseyi S1 - KT692704 Aphelenchoidinae Aphelenchoides besseyi S12 - KT692708 Aphelenchoidinae Aphelenchoides besseyi S2 - KT692705 Aphelenchoidinae Aphelenchoides bicaudatus HQ231404 - Aphelenchoidinae Aphelenchoides bicaudatus AChoBic AY284643 - Aphelenchoidinae Aphelenchoides bicaudatus APMTn_1 JN887885 - Aphelenchoidinae Aphelenchoides bicaudatus APWKs JN887884 - Aphelenchoidinae Aphelenchoides blastophthorus AChoBla AY284643 - Aphelenchoidinae Aphelenchoides fragariae AB067755 - Aphelenchoidinae Aphelenchoides fragariae 1 - KT692710 Aphelenchoidinae Aphelenchoides fragariae 11 - KP835684 Aphelenchoidinae Aphelenchoides fragariae 12 - KP835685 Aphelenchoidinae Aphelenchoides fragariae 13 - KP835686 Aphelenchoidinae Aphelenchoides fragariae 14 - KP835687 Aphelenchoidinae Aphelenchoides fragariae 15 - KP835683 Aphelenchoidinae Aphelenchoides fragariae 2 - KT692711 Aphelenchoidinae Aphelenchoides fragariae 3 - KT692712 Aphelenchoidinae Aphelenchoides fragariae 399 EU325681 EU325684 Aphelenchoidinae Aphelenchoides fragariae 4 - KT261769 Aphelenchoidinae Aphelenchoides fragariae 5 - KT261770 Aphelenchoidinae Aphelenchoides fragariae A_frag_ND1 KX356708 KX356778 Aphelenchoidinae Aphelenchoides fragariae A_frag_ND2 KX356709 KX356779 Aphelenchoidinae Aphelenchoides fragariae AChoFra1 AY284645 - Aphelenchoidinae Aphelenchoides fragariae AfBotSad DQ901551 - Aphelenchoidinae Aphelenchoides fragariae CA21_USA - DQ328683 Aphelenchoidinae Aphelenchoides fragariae Elsanta KT964600 - Aphelenchoidinae Aphelenchoides fragariae Gent AJ966475 - Aphelenchoidinae Aphelenchoides fuchsi KT003986 KT003987 Aphelenchoidinae Aphelenchoides fujianensis Asp4993 KY769067 KY769082 Aphelenchoidinae Aphelenchoides fujianensis DJ01 KT692663 KT692683 Aphelenchoidinae Aphelenchoides fujianensis DJ02 KT692664 KT692684 Aphelenchoidinae Aphelenchoides fujianensis DJ03 KT692665 KT692685 Aphelenchoidinae Aphelenchoides fujianensis DJ04 KT692666 KT692686 Aphelenchoidinae Aphelenchoides fujianensis DJ05F KT692667 KT692687 Aphelenchoidinae Aphelenchoides fujianensis DJ06F KT692668 KT692688 Aphelenchoidinae Aphelenchoides fujianensis DJ07F KT692670 KT692691 Aphelenchoidinae Aphelenchoides fujianensis DJ08 KT692672 KT692692 Aphelenchoidinae Aphelenchoides fujianensis DJ10 KT692673 KT692693 Aphelenchoidinae

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Table 7-S1 (continued) Aphelenchoides fujianensis DJ12F KT692674 KT692695 Aphelenchoidinae Aphelenchoides fujianensis DJ13 KT692676 KT692697 Aphelenchoidinae Aphelenchoides fujianensis DJ14 KT692677 KT692698 Aphelenchoidinae Aphelenchoides fujianensis DJ15F KT692678 KT692699 Aphelenchoidinae Aphelenchoides fujianensis DJ16 KT692680 KT692701 Aphelenchoidinae Aphelenchoides fujianensis DJ17 KT692681 KT692702 Aphelenchoidinae Aphelenchoides fujianensis XP1 FJ520227 - Aphelenchoidinae Aphelenchoides gorganensis KX357651 KX357652 Aphelenchoidinae Aphelenchoides heidelbergi - KT884898 Aphelenchoidinae Aphelenchoides huntensis KR864863 KR864862 Aphelenchoidinae Aphelenchoides iranicus KU565874 KU565873 Aphelenchoidinae Aphelenchoides macronucleatus FJ235883 - Aphelenchoidinae Aphelenchoides macronucleatus 12Asp KY689016 KX356748 Aphelenchoidinae Aphelenchoides macronucleatus Asp6266 KY769068 KY769083 Aphelenchoidinae Aphelenchoides pannocaduatus Lpanno KX356747 KX356838 Aphelenchoidinae Aphelenchoides paradalianensis HR3 GU337993 - Aphelenchoidinae Aphelenchoides paraxui KU738609 KU738610 Aphelenchoidinae Aphelenchoides ritzemabosi 1 - KT692713 Aphelenchoidinae Aphelenchoides ritzemabosi 27957 - KX119135 Aphelenchoidinae Aphelenchoides ritzemabosi 28205 - KX119133 Aphelenchoidinae Aphelenchoides ritzemabosi AchoRit1 JQ957882 - Aphelenchoidinae Aphelenchoides ritzemabosi AchoRit2 JQ957881 - Aphelenchoidinae Aphelenchoides ritzemabosi ArBotSad DQ901554 - Aphelenchoidinae Aphelenchoides ritzemabosi G033 - KX356837 Aphelenchoidinae Aphelenchoides ritzemabosi W10_1 - KR261601 Aphelenchoidinae Aphelenchoides ritzemabosi W10_2 - KR261602 Aphelenchoidinae Aphelenchoides rotundicaudatus KF772858 KF772859 Aphelenchoidinae Aphelenchoides sacchari 10Asp KY689014 - Aphelenchoidinae Aphelenchoides saprophilus 2140 FJ040408 - Aphelenchoidinae Aphelenchoides sp. 11Asp KY689015 KY689008 Aphelenchoidinae Aphelenchoides sp. 13Asp KY689017 KX356749 Aphelenchoidinae Aphelenchoides sp. 14Asp KY689018 KY689009 Aphelenchoidinae Aphelenchoides sp. 15Asp KY689019 KX356750 Aphelenchoidinae Aphelenchoides sp. 16Asp KY689020 KX356751 Aphelenchoidinae Aphelenchoides sp. 17Asp KY689021 KX356752 Aphelenchoidinae Aphelenchoides sp. 18Asp KY769058 KY769072 Aphelenchoidinae Aphelenchoides sp. 19Asp KY769059 KY769073 Aphelenchoidinae Aphelenchoides sp. Ap007 KX356715 KX356789 Aphelenchoidinae Aphelenchoides sp. Ap025 - KX356794 Aphelenchoidinae Aphelenchoides sp. Ap026 - KX356795 Aphelenchoidinae Aphelenchoides sp. Ap027 KX356719 KX356796 Aphelenchoidinae Aphelenchoides sp. Ap046 - KX356802 Aphelenchoidinae Aphelenchoides sp. Ap108 KX356734 KX356818 Aphelenchoidinae Aphelenchoides sp. Asp1764 KY769063 KY769078 Aphelenchoidinae Aphelenchoides sp. Asp2241 KY769064 KY769079 Aphelenchoidinae Aphelenchoides sp. Asp25 KY769062 KY769077 Aphelenchoidinae

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Table 7-S1 (continued) Aphelenchoides sp. Asp2890 - KY769080 Aphelenchoidinae Aphelenchoides sp. Asp3187 KY769065 KY769081 Aphelenchoidinae Aphelenchoides sp. Asp3912 KY769066 - Aphelenchoidinae Aphelenchoides sp. Asp6753 KY769069 KY769084 Aphelenchoidinae Aphelenchoides sp. Asp75747 KY769071 KY769086 Aphelenchoidinae Aphelenchoides sp. Asp7763 KY769070 KY769085 Aphelenchoidinae Aphelenchoides sp. AspDLDD - KY769087 Aphelenchoidinae Aphelenchoides sp. AspNJDH - KY769088 Aphelenchoidinae Aphelenchoides sp. AspX KY769061 KY769076 Aphelenchoidinae Aphelenchoides sp. 8Asp KY689013 KY689006 Aphelenchoidinae Aphelenchoides sp. 1_DSDJ_2016_S6 - KT692706 Aphelenchoidinae Aphelenchoides sp. 2_DSDJ_2016_S9 - KT692707 Aphelenchoidinae Aphelenchoides sp. 2126 FJ040409 - Aphelenchoidinae Aphelenchoides sp. 2130 FJ040410 - Aphelenchoidinae Aphelenchoides sp. 2134 FJ040411 - Aphelenchoidinae Aphelenchoides sp. 2137 FJ040412 - Aphelenchoidinae Aphelenchoides sp. 3_DSDJ_2016_S10_3 - KT692709 Aphelenchoidinae Aphelenchoides sp. 9Asp - KY689007 Aphelenchoidinae Aphelenchoides sp. Ap_A AB661626 - Aphelenchoidinae Aphelenchoides sp. Be GU337995 - Aphelenchoidinae Aphelenchoides sp. BE1 KY963614 KY963618 Aphelenchoidinae Aphelenchoides sp. BE2 KY963615 KY963619 Aphelenchoidinae Aphelenchoides sp. CA22 - DQ328682 Aphelenchoidinae Aphelenchoides sp. E2122 KY963616 KY963620 Aphelenchoidinae Aphelenchoides sp. E2133_2 KY963617 KY963621 Aphelenchoidinae Aphelenchoides sp. E9605 KX356740 KX356780 Aphelenchoidinae Aphelenchoides sp. G022 - KX356832 Aphelenchoidinae Aphelenchoides sp. H1_WY_2008 EU287591 - Aphelenchoidinae Aphelenchoides sp. HB GU337999 - Aphelenchoidinae Aphelenchoides sp. JB011 DQ901550 - Aphelenchoidinae Aphelenchoides sp. JB012 DQ901553 - Aphelenchoidinae Aphelenchoides sp. JH_2004_AChoSp1 AY284646 - Aphelenchoidinae Aphelenchoides sp. K1_WY_2008 EU287589 EU287587 Aphelenchoidinae Aphelenchoides sp. KP GU337994 - Aphelenchoidinae Aphelenchoides sp. N4 LC128708 - Aphelenchoidinae Aphelenchoides sp. SAS_2006 DQ901552 - Aphelenchoidinae Aphelenchoides sp. st1_30 AB630998 - Aphelenchoidinae Aphelenchoides sp. TG102006 - EU084037 Aphelenchoidinae Aphelenchoides sp. US01 GU337998 - Aphelenchoidinae Aphelenchoides sp. YN GU337996 - Aphelenchoidinae Aphelenchoides stammeri AB368535 AM396582 Aphelenchoidinae Aphelenchoides stellatus - KF638651 Aphelenchoidinae Aphelenchoides subtenius F9165 KX356710 KX356778 Aphelenchoidinae Aphelenchoides unisexus 4Asp KY689010 - Aphelenchoidinae Aphelenchoides unisexus 5Asp KY689011 - Aphelenchoidinae Aphelenchoides unisexus 6Asp KY689012 KY689005 Aphelenchoidinae

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Table 7-S1 (continued) Aphelenchoides varicaudatus Nxy17 - HQ283353 Aphelenchoidinae Aphelenchoides xui FJ643487 FJ643488 Aphelenchoidinae Aphelenchoides xylocopae - AB434933 Aphelenchoidinae Aphelenchus avenae 33SR JQ348399 JQ348400 Aphelenchinae Aphelenchus sp. 12-6370 KF032031 KF032032 Aphelenchoidinae Aprutides guidetti KJ636424 - Seinurinae Bursaphelenchus abietinus AY508011 AY508074 Parasitaphelenchinae Bursaphelenchus abruptus AY508010 AY508073 Parasitaphelenchinae Bursaphelenchus africanus africanus JF317266 AM397024 Parasitaphelenchinae Bursaphelenchus africanus rossicus - AM623784 Parasitaphelenchinae Bursaphelenchus anamurius - FJ643489 Parasitaphelenchinae Bursaphelenchus anatolius AY508025 AY508093 Parasitaphelenchinae Bursaphelenchus andrassyi KF164829 KF164833 Parasitaphelenchinae Bursaphelenchus antoniae AM279709 AM279710 Parasitaphelenchinae Bursaphelenchus arthuri AM397010 AM396564 Parasitaphelenchinae Bursaphelenchus arthuroides HQ599188 HQ599190 Parasitaphelenchinae Bursaphelenchus borealis AY508012 AY508075 Parasitaphelenchinae Bursaphelenchus braaschae GQ845409 GQ845408 Parasitaphelenchinae Bursaphelenchus burgermeisteri JF317267 EU159109 Parasitaphelenchinae Bursaphelenchus chengi KT599480 EU107359 Parasitaphelenchinae Bursaphelenchus clavicaudatus AB299221 AB299222 Parasitaphelenchinae Bursaphelenchus cocophilus AY509153 AY508076 Parasitaphelenchinae Bursaphelenchus conicaudatus AB067757 AM396565 Parasitaphelenchinae Bursaphelenchus corneolus corneolus JQ765873 JQ765871 Parasitaphelenchinae Bursaphelenchus corneolus taiwanensis HQ407406 HQ407405 Parasitaphelenchinae Bursaphelenchus debrae - EF488813 Parasitaphelenchinae Bursaphelenchus doui AB299224 AM396567 Parasitaphelenchinae Bursaphelenchus eggersi AY508013 AY508078 Parasitaphelenchinae Bursaphelenchus eremus - AM396568 Parasitaphelenchinae Bursaphelenchus fagi - JX683686 Parasitaphelenchinae Bursaphelenchus firmae AB650015 AB650014 Parasitaphelenchinae Bursaphelenchus fraudulentus AY508015 AY508081 Parasitaphelenchinae Bursaphelenchus fungivorus AY508016 AY508082 Parasitaphelenchinae Bursaphelenchus gerberae AY508024 AY508092 Parasitaphelenchinae Bursaphelenchus gillanii KJ653442 KJ653443 Parasitaphelenchinae Bursaphelenchus hellenicus AY508017 AY508083 Parasitaphelenchinae Bursaphelenchus hildegardae AM397013 AM396569 Parasitaphelenchinae Bursaphelenchus hofmanni AY508018 AY508084 Parasitaphelenchinae Bursaphelenchus hylobianum AY508019 KT806476 Parasitaphelenchinae Bursaphelenchus kesiyae LC087116 LC087117 Parasitaphelenchinae Bursaphelenchus kevini AY753531 AY753532 Parasitaphelenchinae Bursaphelenchus kiyoharai AB597255 AB597254 Parasitaphelenchinae Bursaphelenchus koreanus JX154585 JX154584 Parasitaphelenchinae Bursaphelenchus luxuriosae AB097864 AM396571 Parasitaphelenchinae Bursaphelenchus macromucronatus - EU256382 Parasitaphelenchinae Bursaphelenchus masseyi - JQ287495 Parasitaphelenchinae

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Table 7-S1 (continued) Bursaphelenchus mazandaranense JN153102 JN153103 Parasitaphelenchinae Bursaphelenchus mucronatus AY508023 AM396573 Parasitaphelenchinae Bursaphelenchus niphades AB849465 AB849475 Parasitaphelenchinae Bursaphelenchus obeche - EU159108 Parasitaphelenchinae Bursaphelenchus okinawaensis AB358983 AB358982 Parasitaphelenchinae Bursaphelenchus osumiana AB918706 AB918707 Parasitaphelenchinae Bursaphelenchus paraburgeri HQ727727 HQ727725 Parasitaphelenchinae Bursaphelenchus paracorneolus AY508027 AY508095 Parasitaphelenchinae Bursaphelenchus paraluxuriosae JF966206 JF966204 Parasitaphelenchinae Bursaphelenchus parantoniae KT223041 KT223042 Parasitaphelenchinae Bursaphelenchus paraparvispicularis GQ421483 GQ429010 Parasitaphelenchinae Bursaphelenchus parapinasteri KT878515 KT878516 Parasitaphelenchinae Bursaphelenchus parathailandae JN377724 JN377722 Parasitaphelenchinae Bursaphelenchus parvispicularis AB218829 AB368537 Parasitaphelenchinae Bursaphelenchus penai AB901293 AB901292 Parasitaphelenchinae Bursaphelenchus piceae KT315781 KF772174 Parasitaphelenchinae Bursaphelenchus pinasteri AM397016 AM396574 Parasitaphelenchinae Bursaphelenchus platzeri AY508026 AY508094 Parasitaphelenchinae Bursaphelenchus poligraphi AY508028 AY508096 Parasitaphelenchinae Bursaphelenchus populi HQ699855 FJ998281 Parasitaphelenchinae Bursaphelenchus posterovulvus KF314804 KF314807 Parasitaphelenchinae Bursaphelenchus rainulfi AM397017 AM396575 Parasitaphelenchinae Bursaphelenchus rufipennis AB368529 AB368530 Parasitaphelenchinae Bursaphelenchus sakishimanus LC027461 LC027462 Parasitaphelenchinae Bursaphelenchus Saudi KT806480 KT806482 Parasitaphelenchinae Bursaphelenchus seani AY508029 AY508098 Parasitaphelenchinae Bursaphelenchus sexdentati AY508031 AY508103 Parasitaphelenchinae Bursaphelenchus sinensis AB232162 EU752257 Parasitaphelenchinae Bursaphelenchus singaporensis AM397018 AM396576 Parasitaphelenchinae Bursaphelenchus sycophilus AB901291 AB901290 Parasitaphelenchinae Bursaphelenchus tadamiensis AB635399 AB635398 Parasitaphelenchinae Bursaphelenchus thailandae AM397019 AM396577 Parasitaphelenchinae Bursaphelenchus tiliae - KF736955 Parasitaphelenchinae Bursaphelenchus tokyoensis AB430445 AB430446 Parasitaphelenchinae Bursaphelenchus trypophloei - FJ998283 Parasitaphelenchinae Bursaphelenchus tusciae AY508033 AY508104 Parasitaphelenchinae Bursaphelenchus ulmophilus KR011752 KP331049 Parasitaphelenchinae Bursaphelenchus vallesianus AM397020 AM396578 Parasitaphelenchinae Bursaphelenchus willibaldi AM397021 AM396579 Parasitaphelenchinae Bursaphelenchus xylophilus AM397022 AM396580 Parasitaphelenchinae Bursaphelenchus yongensis AM397023 AM396581 Parasitaphelenchinae Cryptaphelenchus sp. K2_WY_2008 EU287588 EU287596 Ektaphelenchinae Cryptaphelenchus sp. KER - KT895255 Ektaphelenchinae Cryptaphelenchus sp. NK2010 - AB597985 Ektaphelenchinae Cryptaphelenchus sp. SG2014 KJ705087 - Ektaphelenchinae Devibursaphelenchus eproctatus JN122012 - Ektaphelenchinae

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Table 7-S1 (continued) Devibursaphelenchus lini AM397014 - Ektaphelenchinae Devibursaphelenchus sp. KC154092 - Ektaphelenchinae Devibursaphelenchus teratospicularis KC148531 - Ektaphelenchinae Ektaphelenchoides andrassyi - KM272330 Ektaphelenchinae Ektaphelenchoides andrassyi Kch4 - JQ446375 Ektaphelenchinae Ektaphelenchoides caspiensis - KM272329 Ektaphelenchinae Ektaphelenchoides compsi - DQ257625 Ektaphelenchinae Ektaphelenchoides fuchsia - KJ190832 Ektaphelenchinae Ektaphelenchoides hunti - JN714466 Ektaphelenchinae Ektaphelenchoides kelardashtensis Kch2 - JQ446374 Ektaphelenchinae Ektaphelenchoides pini - DQ257623 Ektaphelenchinae Ektaphelenchoides poinari KC881252 - Ektaphelenchinae Ektaphelenchoides poinari A5 - KP313828 Ektaphelenchinae Ektaphelenchoides ruehmi - KF509853 Ektaphelenchinae Ektaphelenchoides sp. FFPRI_SB1 - AB434934 Ektaphelenchinae Ektaphelenchoides spondylis AB849952 - Ektaphelenchinae Ektaphelenchus obtusus AB368532 AB368533 Ektaphelenchinae Ektaphelenchus sp. JH_2012_21748 JX979194 JX979196 Ektaphelenchinae Ektaphelenchus sp. JH_2014 - KM370169 Ektaphelenchinae Ektaphelenchus sp. MP_2016a_ALv - KU373124 Ektaphelenchinae Ektaphelenchus taiwanensis 15287 JX154588 JX154587 Ektaphelenchinae Ficophagus altermacrophylla RGD413 - AB535552 Aphelenchoidinae Ficophagus altermacrophylla RGD75 - AB535534 Aphelenchoidinae Ficophagus altermacrophylla RGD77 - AB535535 Aphelenchoidinae Ficophagus altermacrophylla RGD85 - AB535540 Aphelenchoidinae Ficophagus aureus DQ912922 DQ912925 Aphelenchoidinae Ficophagus benjamina 459 KJ638348 - Aphelenchoidinae Ficophagus benjamina 921 KJ638351 - Aphelenchoidinae Ficophagus benjamina RGD441 - AB535553 Aphelenchoidinae Ficophagus benjamina RGD456 - AB535557 Aphelenchoidinae Ficophagus benjamina RGD459 - AB535558 Aphelenchoidinae Ficophagus benjamina RGD87 - AB535542 Aphelenchoidinae Ficophagus centerae DQ912923 DQ912928 Aphelenchoidinae Ficophagus fleckeri 733 KJ638371 - Aphelenchoidinae Ficophagus laevigatus DQ912920 DQ912926 Aphelenchoidinae Ficophagus microcarpus Sm09 GU229645 GU392234 Aphelenchoidinae Ficophagus sp. 5069 KM817192 KM817193 Aphelenchoidinae Ficophagus sp. ex_Ficus_altissima - Aphelenchoidinae Ficophagus sp. KAD2009_RGD7852 - AB535562 Aphelenchoidinae Ficophagus sp. KAD2009_RGD788 - AB535566 Aphelenchoidinae Ficophagus sp. M_1_RGD448 - AB535556 Aphelenchoidinae Ficophagus sp. M_3_RGD306 - AB535545 Aphelenchoidinae Ficophagus sp. M_3_RGD78516 - AB535563 Aphelenchoidinae Ficophagus sp. M_3_RGD787 - AB535565 Aphelenchoidinae Ficophagus sp. M_8_RGD437 - AB535555 Aphelenchoidinae Ficophagus sp. M_8_RGD80 - AB535538 Aphelenchoidinae

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Table 7-S1 (continued) Ficophagus sp. M_9_RGD442 - AB535554 Aphelenchoidinae Ficophagus sp. WY_2014_914 KJ638349 KJ638372 Aphelenchoidinae Ficophagus sp. WY_2014_915 KJ638350 KJ638373 Aphelenchoidinae Ficophagus sp. WY_2014_931 KJ638354 - Aphelenchoidinae Ficophagus sp. WY_2014_936 KJ638357 - Aphelenchoidinae Ficophagus sp. WY_2014_938 KJ638358 KJ638375 Aphelenchoidinae Ficophagus sp. WY_2014_957 KJ638360 KJ638376 Aphelenchoidinae Ficophagus sp. WY_2014_958 KJ638361 KJ638377 Aphelenchoidinae Ficophagus sp. WY_2014_961 KJ638362 KJ638378 Aphelenchoidinae Ficophagus sp. WY_2014_962 KJ638363 KJ638379 Aphelenchoidinae Ficophagus sp. WY_2014_964 KJ638364 KJ638380 Aphelenchoidinae Ficophagus sp. WY_2014_966 KJ638365 KJ638381 Aphelenchoidinae Ficophagus sp. WY_2014_967 KJ638366 KJ638382 Aphelenchoidinae Ficophagus sp. WY_2014_968 KJ638367 KJ638383 Aphelenchoidinae Ficophagus sp. WY_2014_973 KJ638368 KJ638384 Aphelenchoidinae Ficophagus sp. WY_2014_975 KJ638369 KJ638385 Aphelenchoidinae Ficophagus sp. WY_2014_978 KJ638370 KJ638386 Aphelenchoidinae Ficophagus virens RGD463 - AB535559 Aphelenchoidinae Ficophagus virens RGD786 - AB535564 Aphelenchoidinae Ficophagus virens RGD88 - AB535543 Aphelenchoidinae Ficophagus virens RGD89 - AB535544 Aphelenchoidinae Laimaphelenchus australis - EU287600 Aphelenchoidinae Laimaphelenchus belgradiensis V91 KF881745 KF881746 Aphelenchoidinae Laimaphelenchus deconincki - KF998578 Aphelenchoidinae Laimaphelenchus heidelbergi EU287587 EU287595 Aphelenchoidinae Laimaphelenchus heidelbergi GBA4 - KJ564293 Aphelenchoidinae Laimaphelenchus hyrcanus - KJ567061 Aphelenchoidinae Laimaphelenchus penardi LaimPen1 AY593918 - Aphelenchoidinae Laimaphelenchus penardi LaimPen2 AY593919 - Aphelenchoidinae Laimaphelenchus penardi wb_6 EU306346 - Aphelenchoidinae Laimaphelenchus persicus - JN006987 Aphelenchoidinae Laimaphelenchus preissii EU287590 EU287598 Aphelenchoidinae Laimaphelenchus sp. RA2014 - KJ472144 Aphelenchoidinae Laimaphelenchus sp. RGD636L - AB368539 Aphelenchoidinae Martininema baculum - AB535541 Aphelenchoidinae Martininema sp. KC250362 KC250363 Aphelenchoidinae Martininema sp. KM817189 KM817190 Aphelenchoidinae Martininema guangzhouensis DQ912924 DQ912927 Aphelenchoidinae Noctuidonema sp. RGD820 AB470969 AB470970 Acugutturinae Paraphelenchus acontioides HQ218323 HQ218322 Paraphelenchinae Peraphelenchus orientalis AB786908 AB786909 Entaphelenchinae Pseudaphelenchus jiaae HQ283350 HQ283352 Tylaphelenchinae Pseudaphelenchus scheffrahni AB971163 AB971164 Tylaphelenchinae Pseudaphelenchus sui AB971166 AB971167 Tylaphelenchinae Pseudaphelenchus vindai AB537559 AB537560 Tylaphelenchinae Pseudaphelenchus yukiae AB470971 AB470972 Tylaphelenchinae

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Table 7-S1 (continued) Pseudaphelenchus zhoushanensis KX155843 KX168423 Tylaphelenchinae Robustodorus megadorus KC687094 KC687095 Aphelenchoidinae Ruehmaphelenchus asiaticus - AM269475 Aphelenchoidinae Ruehmaphelenchus digitulus JN377732 JN377730 Aphelenchoidinae Ruehmaphelenchus formaosanus AB808718 AB808719 Aphelenchoidinae Ruehmaphelenchus sp. NK2010c - AB597984 Aphelenchoidinae Ruehmaphelenchus sp. NK202 AB368534 - Aphelenchoidinae Schistonchus benjamina KJ638356 Aphelenchoidinae Schistonchus caprifici 01_26_785 - EU287654 Aphelenchoidinae Schistonchus caprifici 33_03_810 - EU287674 Aphelenchoidinae Schistonchus caprifici 6 GU190763 - Aphelenchoidinae Schistonchus caprifici 7 GU190764 - Aphelenchoidinae Schistonchus caprifici 74_Riva_Marina FN564938 - Aphelenchoidinae Schistonchus caprifici AMSR_4A_827 - EU287689 Aphelenchoidinae Schistonchus caprifici MAR_1_383 - EU287692 Aphelenchoidinae Schistonchus hirtus GQ849472 GQ849473 Aphelenchoidinae Schistonchus macrophylla RGD366 - AB535546 Aphelenchoidinae Schistonchus macrophylla RGD367 - AB535547 Aphelenchoidinae Schistonchus macrophylla RGD368 - AB535548 Aphelenchoidinae Schistonchus macrophylla RGD50 - AB535530 Aphelenchoidinae Schistonchus macrophylla RGD72 - AB535531 Aphelenchoidinae Schistonchus macrophylla RGD73 - AB535532 Aphelenchoidinae Schistonchus macrophylla RGD74 - AB535533 Aphelenchoidinae Schistonchus macrophylla RGD79 - AB535537 Aphelenchoidinae Schistonchus macrophylla RGD82 - AB535539 Aphelenchoidinae Schistonchus sp. WY_2014_369 KJ638347 - Aphelenchoidinae Schistonchus sp. YZ_2010_Sw10 HM151003 - Aphelenchoidinae Schistonchus sp. YZ_M5_RGD731 AB535560 - Aphelenchoidinae Seinura demani SeinDem1 FJ969140 - Seinurinae Seinura demani SeinDem1 JQ957894 - Seinurinae Seinura sp. 1_YA_2016 KU991832 - Seinurinae Seinura sp. Fars - KT355496 Seinurinae Seinura sp. Gilan - KT354242 Seinurinae Seinura sp. Golestan - KT355495 Seinurinae Seinura sp. JH_2004_SeinSp AY284651 - Seinurinae Sheraphelenchus parabrevigulonis KC875226 KC875232 Aphelenchoidinae Sheraphelenchus sucus AB808720 AB808721 Aphelenchoidinae Sheraphelenchus sucus 10984 KC875229 KC894755 Aphelenchoidinae

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Chapter VIII

General discussion and conclusion

195

The objective of present study was to enrich the taxonomic information and update several morphological and molecular aspects of the family Aphelenchoididae. In this research, two new and five unknown species of the genus Aphelenchoides were described by morphological and molecular characterisations (see Chapter II, III and VI). In addition to this, the taxonomic status of Sheraphelenchus and Pseudaphelenchus in Aphelenchoididae was evaluated and clarified (see Chapter IV and V). Furthermore, the concatenated phylogenetic analyses of 18S and 28S D2-D3 (see Chapter VII) showed five major clades in Aphelenchoididae, the relationship between head region characters and phylogenetic results was also discussed, and detailed illustrations of different head regions were presented (see Chapter VII). The major taxonomical, phylogenetic, and ecological aspects of the genera Aphelenchoides, Sheraphelenchus and Pseudaphelenchus are as follows.

Taxonomy and phylogeny in Aphelenchoididae

Aphelenchoides

The phylogenetic analyses of Aphelenchoididae demonstrated a clear paraphyletic relationship of Aphelenchoides with Ficophagus, Martininema, Laimaphelenchus, Schistonchus, Aprutides and Robustodorus, which supports earlier evidence by Esmaeili et al., 2016b, b; Kanzaki et al., 2014c; Kanzaki & Giblin-Davis 2012; Rybarczyk-Mydłowska et al., 2012; and Jesus et al., 2016. However, most of the morphological characteristics were not in accordance with phylogenetic clades. Only the tail and tail-terminus shapes of Aphelenchoides species appeared to, largely agreeing with natural groups (Sánchez-Monge 2016).

Sheraphelenchus

Our phylogenetic analyses revealed that S. parabrevigulonis and S. sucus clustered into an independent clade and imbedded in Bursaphelenchus species, indicating that Sheraphelenchus is a monophyletic group that may share a recent common ancestor with Bursaphelenchus. Based upon their molecular phylogeny, Kanzaki & Tanaka (2013) considered that Sheraphelenchus was probably a junior synonym of Bursaphelenchus. Among the species of

196

Bursaphelenchus, B. kiyoharai and B. posterovulvus showed a close similarity with Sheraphelenchus species regarding tail shape and posteriorly located vulva, respectively. It may demonstrate that some apomorphic characters can appear several times in different clades among Bursaphelenchus. Hunt (1993) characterised Sheraphelenchus as having well-developed stylet and lacking basal knobs or swellings. However, through the high-resolution photomicrographs in this study and Kanzaki & Tanaka. (2013), a stylet with slight basal swellings was clearly observed in Sheraphelenchus.

Pseudaphelenchus

Pseudaphelenchus and Tylaphelenchus share several morphological characters, and clustered together as a basal clade in the phylogenetic analysis of Aphelenchoididae. To date only one Tylaphelenchus species was molecularly characterized i.e., T. jiaae. Based on the molecular data obtained through this sole species of Tylaphelenchus, Kanzaki et al., (2014c) erected a new subfamily Tylaphelenchinae including Tylaphelenchus and Pseudaphelenchus. In this study, we reviewed the morphological and molecular characters of T. jiaae and propose to transfer this species to Pseudaphelenchus, making the existence and status of Tylaphelenchinae questionable according to our phylogenetic analysis.

Ecology diversity in Aphelenchoididae

Aphelenchoides

Bark and wood samples are considered as rich substrates for Aphelenchoides species; several new species, namely A. huntensis (Esmaeili et al., 2016b), A. fuchsi (Esmaeili et al., 2016c), A. iranicus (Golhasan et al. 2016) , A. paraxui (Esmaeili et al., 2017a) and A. eldaricus (Esmaeili et al., 2017b) were recently described from this kind of samples. In this study, the Aphelenchoides species which were also isolated from wood samples and pine trees (see Chapter VI) showed a remarkably high sequences variation in different populations, suggesting that these populations belong to different species. Not only the populations from bark and wood samples, but also the populations from different moss, fungi and soil have are molecularly highly diverse (Sánchez-Monge 2016). Furthermore, it is rare to find the same

197 species in different habitats e.g. Aphelenchoides sp. 12Asp (isolated from Pinus thunbergii) and Aphelenchoides sp. Asp6266 (isolated from Acer palmatum). However, within Clade 2b, the same Aphelenchoides species could be found in different habitats e.g. Aphelenchoides besseyi could be found in wood packaging and different soil and plant tissues (Sánchez-Monge et al., 2015).

Sheraphelenchus

Sheraphelenchus species were presumed to be associated with nitidulid beetles, such as S. brevigulonis that was isolated from the necrotic tissues of Cenangium and Ceratocystis cankers of aspens associated with Epurea sp. (Massey & Hinds, 1970) and S. entomophagus from Carpophilus mutilates and Urophorus humeralis (Nickle, 1970). While S. sucus presumed to be associated with nitidulid beetles as it was found from the sap flow of Quercus trees (Kanzaki & Tanaka, 2013). In this study, S. parabrevigulonis was isolated from pine wood packaging material imported from Italy and it is assumed that the pine wood could have been infested by pine bark beetles, the eggs of which can be predated by a type of nitidulid beetle, Mimemodes japonus (Kishi, 1970). Similarly, the redescribed isolate 10984 of S. sucus was obtained from onion bulbs imported from South Korea, which may have decomposed, thereby producing the volatiles that are known to attract nitidulid sap beetles (Nout & Bartelt, 1998).

Pseudaphelenchus

Until 2016, all known Pseudaphelenchus species were isolated from termites (Kanzaki et al., 2009a, 2010, 2014), however P. zhoushanensis was isolated from Pinus thunbergii wood samples (Fang et al., 2016). No termites were observed; only scolytid (bark beetles) tunnels were visible on the dead P. thunbergii tree. This is the first report of Pseudaphelenchus species isolated from P. thunbergii and reared on B. fuckeliana successfully. Although the detailed life cycles of Pseudaphelenchus are still unknown, possibly they may have a strategy of switching hosts via insects, such as from a dead pine tree to another hosts with fresh fungi, a life cycle similar to Bursaphelenchus (Hunt, 1993). In the phylogenetic tree (see chapter VIII), Pseudaphelenchus represent a basal clade (Clade 1) in Aphelenchoididae, and also

198 other clades (Clade 2a, 2b, 3, 4, 5) are represented by different genera and species with a close relationship with insects, thereby indicating that the common ancestor of Aphelenchoididae is possibly associated with insects and that co-evolution with insects is possible (Siddiqi 2000).

Mapping head region characteristics in Aphelenchoididae

Five major clades of Aphelenchoididae were obtained by concatenated phylogenetic analyses of 18S rDNA and D2-D3 28S rDNA gene sequences, a total 404 of taxa were used in the analysis (see chapter VIII). Our tree presents a similar topology structure as obtained by Kanzaki & Giblin-Davies (2012). Hooper & Clark (1980) suggested that Aphelenchoididae has a relatively primitive arrangement of the head region, based on the widely spaced amphidial openings, prominent cephalic papillae and the position of the inner labial papillae around the stomatal aperture. Our new evidence and data mining of the head region characteristics in Aphelenchoididae indicates a high variation in the oral apertures, inner labial sensilla, outer labial sensilla, cephalic sensilla, amphids apertures and annulations of head region. However, the head patterns are not informative to support phylogenetic clades, also a high divergence in cephalic characters was found within genera. Yet, head patterns are informative to diagnose some closely related species of Aphelenchoides, especially for Aphelenchoides besseyi and A. fujianensis. Aphelenchoides besseyi is more likely a complex of species based on molecular and ecological data (Sánchez-Monge 2016). Furthermore, the phylogenetic divergence of the “Rice” and “Beans” groups, already supported by limited morphological and molecular evidence, is now also supported by the head patterns characteristics. Hence, the head region characteristics of Aphelenchoididae appear to be informative taxonomic problems and need to be considered in further studies.

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List of Publications

1. Fang, Y., Gu, J., Wang, X. & Li, H. (2014). Description of Aphelenchoides stellatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from Japan. Nematology 16, 621-630.

2. Fang, Y., Wang, X., Gu, J. & Li, H. (2014). Description of Aphelenchoides rotundicaudatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from South Korea. Nematology 16, 751-760.

3. Fang, Y., Gu, J., Wang, X., Wang, J. & Li, H. (2015). Description of Sheraphelenchus parabrevigulonis n. sp. (Nematoda: Aphelenchoididae) in pine wood packaging from Italy and redescription of S. sucus in onion bulbs from South Korea, isolated at Ningbo, China. Nematology 17, 213-229.

4. Fang, Y., Li, H., Maria, M. & Bert, W. (2016). Description of Pseudaphelenchus zhoushanensis n. sp. (Tylenchina: Aphelenchoididae) found in the wood of Pinus thunbergii at Zhoushan Islands, Zhejiang Province, China. Nematology 18, 1151-1164.

5. Maria, M., Fang, Y., He, J., Gu, J.F. & Li, H.M. (2015). Bursaphelenchus parantoniae n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from Belgium. Nematology 17, 1141-1152.

6. Esmaeili, M., Fang, Y., Li, H. & Heydari, R. (2016). Description of Aphelenchoides huntensis sp. n. (Nematoda: Aphelenchoididae) isolated from Pinus sylvestris in western Iran. Nematology 18, 357-366.

7. Maria, M., Fang, Y., Gu, J. & Li, H. (2016). Redescription of Bursaphelenchus parapinasteri (Tylenchina: Aphelenchoididae) isolated from Pinus thunbergii in China with a key to the hofmanni-group. Nematology 18, 933-947.

8. Esmaeili, M., Heydari, R., Fang, Y. & Li, H.M. (2017). Molecular and morphological characterisation of Aphelenchoides paraxui n. sp. (Nematoda: Aphelenchoididae) isolated from Quercus brantii, in western Iran. European Journal of Plant Pathology 2017, 227

625-637.

9. Sánchez-Monge, A., Janssen, T., Fang, Y., Couvreur, M., Karssen, G. & Bert, W. (2017). mtCOI successfully diagnoses the four main plant-parasitic Aphelenchoides species (Nematoda: Aphelenchoididae) and supports a multiple origin of plant-parasitism in this paraphyletic genus. European Journal of Plant Pathology 148, 853-866.

10. Gu, J., Maria, M., Fang, Y., He, J., Braasch, H. & Li, H. (2016). Bursaphelenchus saudi n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from Saudi Arabia. Nematology 18, 475-488.

11. Maria, M, Gu, J., Fang, Y., He, J., Castillo, P. & Li, H. (2017). Radopholoides japonicus n. sp. (Nematoda: Pratylenchidae) found in rhizosphere soil associated with Podocarpus macrophyllus from Japan. Nematology 19, Online.

12. Esmaeili, M., Heydari, R., Taheri, Z., Fang, Y. & Li, H. (2016). Description of Cryptaphelenchus iranicus n. sp. (Nematoda: Ektaphelenchinae) recovered from bark samples of Pinus nigra from Iran. Zootaxa 4139, 117-127.

13. Maria, M., Gu, J., Tomalak, M., Fang, Y. & Li, H. (2017). Description of Ruehmaphelenchus quercophilus n. sp. (Nematoda: Aphelenchoididae) from dying oak, Quercus robur, in Poland. Nematology 19, Online.

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