Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

Abstract

The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.

Introduction

The spider mite sub-family Tetranychinae includes some pests that cause serious economic losses throughout the world [1], [2], [3]. The family consists of more than 1,200 species, some of which have a wide host range, whereas others are highly host-specific [4], [5]. For example, Tetranychus urticae Koch, Panonychus citri (McGregor) and Oligonychus coffeae (Nietner), have an especially strong effect on agricultural and horticultural crops, and they are polyphagous. However, these genera also include mono-, oligophagous species, such as Tetranychus bambusae Wang & Ma, Panonychus bambusicola Ehara & Gotoh, Oligonychus orthius Rimando, Oligonychus modestus (Banks) and Oligonychus rubicundus Ehara which inhabit only gramineous plants.

Although exact species identification is the first step in any biological study, spider mites are difficult to distinguish by morphological characters alone because of their small size (<0.5 mm) and limited number of diagnostic characters [6], [7], [8]. Therefore, the use of DNA-based methods to identify species has increasingly been used for some genera of the Tetranychinae. For example, Navajas and Boursot [9] showed that T. urticae and Tetranychus turkestani Ugarov & Nikolskii, which are very closely related species, can be identified by using the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal RNA (rRNA) genes. More recently, Matsuda et al. [10], [11] revealed that almost all species of Japanese Oligonychus (17 of 18 species) and all species of Tetranychus (13 species) can be identified by using the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA.

Despite recent advances in DNA-based methods for identifying spider mites, most phylogenetic relationships of sub-families, tribes and genera of the Tetranychinae remain poorly understood, as is reflected by the low support values for most nodes of the phylogenetic trees. However, phylogenetic trees clearly show that the genus Oligonychus is polyphyletic. Navajas et al. [12] and Ros and Breeuwer [13] analyzed the phylogeny of Tetranychinae including three Oligonychus species (Oligonychus ununguis (Jacobi), Oligonychus platani (McGregor) and Oligonychus gossypii (Zacher)) using the COI gene. Although these three species have the same empodium shape, O. gossypii, whose aedeagus curves dorsally, can be easily distinguished from O. ununguis and O. platani whose aedeagi curve ventrally. In the phylogenetic trees of these two studies, O. gossypii clustered more closely with Tetranychus species whose aedeagi also curve dorsally, while O. ununguis and O. platani formed a separate group. Polyphyly in the genus Oligonychus was also reported in the ITS2 region [14].

The unresolved phylogeny among the taxa of the sub-family Tetranychinae based on the COI sequences is probably due to the strongly biased nucleotide composition and the saturation at the third codon positions [13]. Because both the 18S and 28S rRNA genes evolve more slowly and are made up of conserved regions and divergent domains [15], these genes have been used for phylogenetic analyses of higher taxonomic relationships (from “phyla” to “classes” within Ecdysozoa) [16], [17]. In resolving tick genera (Acari: Ixodida), combining the 18S and 28S rRNA genes provided more detailed relationships than did the 18S gene alone [18], [19]. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae. Then, we compared the trees based on the 18S and 28S genes with the tree based on the mitochondrial COI gene (618 bp). Another problem in previous studies [12], [13], [14] was that only 16 to 25 species were used for the phylogenetic analyses. Limited taxon sampling can seriously influence the resulting phylogenetic inferences (for reviews, see [20], [21], [22]). Therefore, to assess the phylogenetic relationships among tribes and genera of the sub-family Tetranychinae, we examined a total of 88 strains (15 genera and 4 tribes) most of which were from Japan.

Results

Mitochondrial COI gene

We obtained the COI sequences of 38 strains determined in this study (Table 1) and 30 strains from previously published data [10], [11]. The COI sequences contained no insertions or deletions. After alignment, the COI fragment had 618 nucleotides, of which 282 were parsimony-informative sites (File S1). The AT contents of the COI sequences of the tetranychid mites were very high (75.5%), especially at the 3rd codon position (93.0%). Chi-square tests revealed no significant heterogeneity in the first and second codon positions of the COI sequences, but significant heterogeneity at third codon positions (Figure 1). Similar high AT contents have been observed in previous studies of tetranychid mites [10], [11], [12], [13].

Table 1. Classification and sources of Tetranychid mites used in this study.

Sub-family	Tribe	Genus	Species	Date	Locality	Host plant	Voucher specimen no.a	Accession no.
								COI	18S	28S
Bryobiinae	Bryobiini	Bryobia	B. eharai Pritchard & Keifer	Sept. 11, 2012	Ibaraki, Japan	Chrysanthemum morifolium	0612	–	AB926227	AB926318
			B. praetiosa Koch	July 27, 2008	Hokkaido, Japan	Trifolium repens	0609	AB981203	AB926228	AB926319
	Petrobiini	Petrobia	P. latens (Müller)	Mar. 30, 2012	Tokushima, Japan	Daucus carota	0482	AB981204	AB926229	AB926320
		Tetranychina	T. harti (Ewing)	June 11, 2012	Ibaraki, Japan	Oxalis corniculata	0602	–	AB926230	AB926321
Tetranychinae	Eurytetranychini	Eurytetranychoides	E. japonicus (Ehara)	Sept. 22, 2010	Tokyo, Japan	Lithocarpus edulis	0493	AB981205	AB926231	AB926322
		Eutetranychus	E. africanus (Tucker)	June 30, 2008	Taichung, Taiwan	Pueraria montana	0377	–	AB926232	AB926323
		Aponychus	A. corpuzae Rimando	Apr. 10, 2001	Ibaraki, Japan	Sasa senanensis	0607	AB981206	AB926233	AB926324
			A. firmianae (Ma & Yuan)	Aug. 7, 2010	Ibaraki, Japan	Firmiana simplex	0405	–	AB926234	AB926325
	Tetranychini	Panonychus	P. bambusicola Ehara & Gotoh	June 4, 1989	Hokkaido, Japan	Sasa senanensis	0606	AB981207	AB926235	AB926326
			P. caglei Mellot	Aug. 19, 2009	Okinawa, Japan	Trichosanthes pilosa	0611	–	AB926236	AB926327
			P. citri (McGregor)	May 6, 1993	Ibaraki, Japan	Ilex crenata	0226	AB981208	AB926237	AB926328
			P. elongatus Manson	July 27, 2010	Hangzhou, China	Broussonetia papyrifera	0398	–	AB926238	AB926329
			P. mori Yokoyama	Apr. 22, 2007	Hokkaido, Japan	Morus australis	0239	AB981209	AB926239	AB926330
			P. osmanthi Ehara & Gotoh	Nov. 16, 2001	Guilin, China	Osmanthus fragrans	0229	AB981210	AB926240	AB926331
			P. thelytokus Ehara & Gotoh	Aug. 4, 2010	Hokkaido, Japan	Ulmus davidiana	0407	AB981211	AB926241	AB926332
			P. ulmi (Koch)	Aug. 2, 2012	Nagano, Japan	Malus pumila	0603	AB981212	AB926242	AB926333
		Sasanychus	S. akitanus (Ehara)	June 23, 1986	Hokkaido, Japan	Sasa senanensis	0605	AB981213	AB926243	AB926334
			S. pusillus Ehara & Gotoh	July 31, 2012	Hokkaido, Japan	Sasa chartacea	0575	AB981214	AB926244	AB926335
		Schizotetranychus	S. bambusae Reck	Aug. 27, 2011	Chiba, Japan	Phyllostachys edulis	0503	AB981215	AB926245	AB926336
			S. brevisetosus Ehara	Oct. 13, 2011	Kochi, Japan	Quercus glauca	0527	AB981216	AB926246	AB926337
			S. cercidiphylli Ehara	Aug. 3, 2010	Hokkaido, Japan	Cercidiphyllum japonicum	0411	AB981217	AB926247	AB926338
			S. gilvus Ehara & Ohashi	May 22, 2012	Nara, Japan	Quercus gilva	0549	AB981218	AB926248	AB926339
			S. lespedezae Begljarov & Mitrofanov	Aug. 26, 2011	Ibaraki, Japan	Pueraria montana	0515	AB981219	AB926249	AB926340
			S. recki Ehara	Aug. 4, 2010	Hokkaido, Japan	Sasa senanensis	0408	AB981220	AB926250	AB926341
			S. schizopus (Zacher)	June 14, 2010	Tokyo, Japan	Salix integra	0532	AB981221	AB926251	AB926342
			S. shii (Ehara)	June 14, 2010	Tokyo, Japan	Castanopsis sieboldii	0533	AB981222	AB926252	AB926343
		Stigmaeopsis	S. celarius Banks	Aug. 7, 2011	Ibaraki, Japan	Pleioblastus chino	0506	AB981223	AB926253	AB926344
			S. longus (Saito)	June 4, 1989	Hokkaido, Japan	Sasa senanensis	0542	AB981224	AB926254	AB926345
			S. miscanthi (Saito)	Feb. 16, 2009	Nagasaki, Japan	Miscanthus sinensis	0404	AB981225	AB926255	AB926346
			S. saharai Saito & Mori	Aug. 5, 2011	Chiba, Japan	Pleioblastus chino	0501	AB981226	AB926256	AB926347
			S. takahashii Saito & Mori	Oct. 27, 1997	Hokkaido, Japan	Sasa senanensis	0541	AB981227	AB926257	AB926348
		Yezonychus	Y. sapporensis Ehara	Aug. 4, 2010	Hokkaido, Japan	Sasa senanensis	0406	AB981228	AB926258	AB926349
		Eotetranychus	E. asiaticus Ehara	Mar. 19, 2007	Nagasaki, Japan	Citrus reticulata	0546	AB981229	AB926259	AB926350
			E. boreus Ehara	June 3, 2010	Wakayama, Japan	Armeniaca mume	0415	–	AB926260	AB926351
			E. celtis Ehara	Aug. 27, 2011	Chiba, Japan	Aphananthe aspera	0502	AB981230	AB926261	AB926352
			E. cornicola Ehara	Aug. 5, 2011	Chiba, Japan	Cornus controversa	0498	AB981231	AB926262	AB926353
			E. dissectus Ehara	Aug. 3, 2010	Hokkaido, Japan	Acer pictum	0412	AB981232	AB926263	AB926354
			E. nomurai Ehara	Aug. 20, 2011	Ibaraki, Japan	Celtis sinensis	0514	AB981233	AB926264	AB926355
			E. pruni (Oudemans)	Sept. 1, 2012	Ibaraki, Japan	Castanea crenata	0562	–	AB926265	AB926356
			E. querci Reeves	Aug. 3, 2010	Hokkaido, Japan	Tilia japonica	0403	–	AB926266	AB926357
			E. quercifoliae Ehara & Gotoh	July 6, 2011	Ibaraki, Japan	Quercus serrata	0507	AB981234	AB926267	AB926358
			E. rubricans Ehara	Sept. 1, 2012	Ibaraki, Japan	Carpinus tschonoskii	0559	–	AB926268	AB926359
			E. smithi Pritchard & Baker	Aug. 14, 2007	Nagasaki, Japan	Rosa multiflora	0545	AB981235	AB926269	AB926360
			E. spectabilis Ehara	Sept. 7, 2011	Hokkaido, Japan	Acer pictum	0524	–	AB926270	AB926361
			E. suginamensis (Yokoyama)	Aug. 26, 2011	Ibaraki, Japan	Morus australis	0517	AB981236	AB926271	AB926362
			E. tiliarium (Hermann)	Aug. 3, 2010	Hokkaido, Japan	Alnus hirsuta	0409	–	AB926272	AB926363
			E. toyoshimai Ehara & Gotoh	Aug. 29, 2011	Iwate, Japan	Magnolia obovata	0519	–	AB926273	AB926364
			E. uchidai Ehara	Aug. 15, 2011	Hokkaido, Japan	Ulmus davidiana	0528	AB981237	AB926274	AB926365
			E. uncatus Garman	Aug. 3, 2010	Hokkaido, Japan	Betula platyphylla	0413	–	AB926275	AB926366
		Oligonychus	O. amiensis Ehara & Gotoh	July 13, 2005	Ibaraki, Japan	Lithocarpus edulis	0116	AB683672	AB926276	AB926367
			O. biharensis (Hirst)	Dec. 21, 2007	Okinawa, Japan	Mangifera indica	0012	AB683678	AB926277	AB926368
			O. camelliae Ehara & Gotoh	May 13, 2000	Fukushima, Japan	Camellia japonica	0082	AB683662	AB926278	AB926369
			O. castaneae Ehara & Gotoh	May 5, 2009	Ibaraki, Japan	Castanea crenata	0297	AB683667	AB926279	AB926370
			O. clavatus (Ehara)	July 28, 2009	Kanagawa, Japan	Pinus thunbergii	0360	AB683654	AB926280	AB926371
			O. coffeae (Nietner)	May 30, 2005	Okinawa, Japan	Mangifera indica	0078	AB683670	AB926281	AB926372
			O. gotohi Ehara	July 1, 2007	Ibaraki, Japan	Lithocarpus edulis	0076	AB683668	AB926282	AB926373
			O. hondoensis (Ehara)	Aug. 22, 2009	Aomori, Japan	Cryptomeria japonica	0376	AB683658	AB926283	AB926374
			O. ilicis (McGregor)	Oct. 30, 2000	Kagoshima, Japan	Camellia sinensis	0081	AB683660	AB926284	AB926375
			O. karamatus (Ehara)	Aug. 27, 2009	Hokkaido, Japan	Larix kaempferi	0358	AB683656	AB926285	AB926376
			O. modestus (Banks)	Sept. 9, 2008	Okinawa, Japan	Digitaria ciliaris	0092	AB683677	AB926286	AB926377
			O. orthius Rimando	July 9, 2009	Okinawa, Japan	Saccharum officinarum	0378	AB683675	AB926287	AB926378
			O. perditus Pritchard & Baker	Sept. 17, 2009	Kanagawa, Japan	Juniperus sp.	0364	AB683665	AB926288	AB926379
			O. pustulosus Ehara	Aug. 22, 2009	Aomori, Japan	Cryptomeria japonica	0363	AB683655	AB926289	AB926380
			O. rubicundus Ehara	Oct. 17, 2008	Kochi, Japan	Miscanthus sinensis	0290	AB683681	AB926290	AB926381
			O. ununguis (Jacobi)	July 27, 2008	Hokkaido, Japan	Cryptomeria japonica	0088	AB683664	AB926291	AB926382
		Amphitetranychus	A. quercivorus (Ehara & Gotoh)	July 9, 2003	Ibaraki, Japan	Quercus crispula	0610	AB981238	AB926292	AB926383
			A. viennensis (Zacher)	Sept. 21, 2010	Tokyo, Japan	Armeniaca vulgaris	0613	AB981239	AB926293	AB926384
		Tetranychus	T. bambusae Wang & Ma	July 5, 2009	Okinawa, Japan	Phyllostachys edulis	0343		AB926294	AB926385
			T. evansi Baker & Pritchard	Nov. 3, 2006	Tokyo, Japan	Solanum nigrum	0210	AB736039	AB926295	AB926386
			T. ezoensis Ehara	Sept. 3, 2008	Ibaraki, Japan	Taxus cuspidata	0281	AB736042	AB926296	AB926387
			T. huhhotensis Ehara, Gotoh & Hong	July 26, 2007	Inner Mongolia Autonomous Region, Mongolia	Zea mays	0201	–	AB926297	AB926388
			T. kanzawai Kishida	May 19, 1993	Shizuoka, Japan	Thea sinensis	0158	AB736043	AB926298	AB926389
			T. lombardinii Baker & Pritchard	July 10, 2008	Durban, South Africa	Erythrina variegata	0381	–	AB926299	AB926390
			T. ludeni Zacher	Oct.17, 1995	Ibaraki, Japan	Solidago virgaurea	0189	AB736051	AB926300	AB926391
			T. macfarlanei Baker & Pritchard	Sept. 30, 2008	Mymensingh, Bangladesh	Dolichos lablab	0389	–	AB926301	AB926392
			T. merganser Boudreaux	Apr. 6, 2007	El Talo, Sonora, Mexico	Cucurbita maxima	0225	–	AB926302	AB926393
			T. misumaiensis Ehara & Gotoh	Aug. 23, 2005	Hokkaido, Japan	Apios sp.	0218	AB736054	AB926303	AB926394
			T. neocaledonicus Andre	May 27, 1998	Tokyo, Japan	Morus australis	0192	AB736055	AB926304	AB926395
			T. okinawanus Ehara	June 19, 2003	Okinawa, Japan	Pueraria montana	0208	AB736058	AB926305	AB926396
			T. parakanzawai Ehara	June 5, 1993	Ibaraki, Japan	Pueraria montana	0155	AB736060	AB926306	AB926397
			T. phaselus Ehara	June 29, 2000	Ibaraki, Japan	Glycine max	0191	AB736066	AB926307	AB926398
			T. piercei McGregor	Dec. 20, 2007	Okinawa, Japan	Cucumis melo	0014	AB736068	AB926308	AB926399
			T. pueraricola Ehara & Gotoh	Oct. 23, 1993	Ibaraki, Japan	Pueraria montana	0203	AB736071	AB926309	AB926400
			T. truncatus Ehara	May 8, 2004	Kyoto, Japan	Solanum nigrum	0195	AB736075	AB926310	AB926401
			T. turkestani Ugarov & Nikolski	Sept. 15, 2007	Hamedan, Iran	Phaseolus vulgaris	0219	AB981240	AB926311	AB926402
			T. urticae Koch (green form)	July 16, 2001	Hokkaido, Japan	Citrullus lanatus	0181	AB736076	AB926312	AB926403
			T. urticae Koch (red form)	Aug. 27, 2001	Nagano, Japan	Dianthus sp.	0171	AB736079	AB926313	AB926404
			T. zeae Ehara, Gotoh & Hong	July 26, 2007	Inner Mongolia Autonomous Region, Mongolia	Zea mays	0202	–	AB926314	AB926405

^aVoucher specimens are preserved at the Laboratory of Applied Entomology and Zoology, Faculty of Agriculture, Ibaraki University under the serial voucher specimen number.

Figure 1. Base compositions of the codons of the mitochondrial COI gene.

(A) All codon positions, (B) 1st codon position, (C) 2nd codon position, (D) 3rd codon position, averaged over all 68 mite strains used in this study. Error bars depict range. Results of the homogeneity test are given for each codon position.

A phylogenetic tree of the sub-family Tetranychinae based on the COI gene is shown in Figure 2. Among the eight genera for which more than two strains were sampled, four genera (Panonychus, Sasanychus, Stigmaeopsis and Amphitetranychus) appear to be monophyletic with >80 bootstrap values, while the other four (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) are polyphyletic. The four monophyletic genera are in clades 8, 3, 5 and 2, respectively (Figure 2). As was observed in previous studies, Oligonychus species whose aedeagus curves ventrally (clade 7) can be easily distinguished from Oligonychus biharensis (Hirst), O. modestus, O. orthius and O. rubicundus whose aedeagi curve dorsally. Although Schizotetranychus and Eotetranychus are scattered across the tree, some species formed well-supported clades. Schizotetranychus bambusae Reck & Schizotetranychus recki Ehara clustered with Sasanychus and Yezonychus species (clade 4). The clade including Schizotetranychus cercidiphylli Ehara, Eotetranychus asiaticus Ehara and Eotetranychus cornicola Ehara are supported with high bootstrap value (clade 6: bootstrap value (BP) = 88). The COI tree also shows monophyly of closely related species that morphologically and molecularly resemble each other, such as P. citri and Panonychus osmanthi Ehara & Gotoh [23], [24] (clade 9) and T. urticae and T. turkestani [9] (clade 1). These results are consistent with the 18S and 28S topologies described below. However, the COI phylogeny was not resolved and the deep-level relationships were especially unresolved, as shown by the low bootstrap values (Figure 2), as was observed in previous studies [12], [13]. The deep-level phylogeny of the sub-family Tetranychinae was also not resolved in the Bayesian tree (data not shown).

Figure 2. Maximum likelihood (ML) phylogenetic tree of the sub-family Tetranychinae based on the mitochondrial COI gene using the GTR Gamma model.

Bootstrap values (>50%) based on 1,000 replications are indicated at nodes. Each operational taxonomic unit is indicated by the voucher specimen no. and scientific name. Black circles with numbers indicate the clade no. which corresponds with the article.

18S and 28S rRNA genes

We determined the 18S and the 5′ end of the 28S rRNA sequences of all 88 strains used in this study (Table 1). The lengths of the 18S sequences obtained were 1,825–1,901 bp. The 18S and 28S sequences contained a number of gaps (insertions and deletions). After alignment and deletion of the ambiguous part of the aligned data, the final length was 1,863 bp, containing 495 parsimony-informative sites. The lengths of the 28S sequences were 646–743 bp, with a final length of 671 bp, containing 201 parsimony-informative sites. The aligned sequences before and after deleting the ambiguous parts are shown in Supporting Information (Files S2–S4). Chi-square tests revealed no significant heterogeneity in the nucleotide composition of the 18S and 28S sequences (Figure 3).

Figure 3. Base compositions of the (A) 18S and (B) 28S rRNA genes, averaged over all 88 mite strains used in this study.

Error bars depict range. Results of the homogeneity test are given for each gene.

Phylogenetic trees based on a single gene were not as well resolved as phylogenetic trees based on the combined 18S and 28S data sets. Therefore, only the combined data set was used for the ML and Bayesian analyses. The 18S and 28S trees suggest that the tribes Bryobiini and Petrobiini of the sub-family Bryobiinae, which were used as outgroups, are both monophyletic (Figures 4A and 5A, clades 22 and 23). Within the Tetranychinae, Clade 15 is composed of species of Eurytetranychini, and clades 12,17 and 20 are composed of species of Tetranychini (Figures 4A and 5A). Among the 10 genera for which more than two strains were sampled, six genera (Bryobia, Aponychus, Panonychus, Sasanychus, Stigmaeopsis and Amphitetranychus), appear to be monophyletic with >95 bootstrap values and 1.00 posterior probabilities, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) are polyphyletic. The monophyletic genera are in clades 22, 14, 5, 7, 17 and 21, respectively (Figures 4A–4D and 5A–5D). Species of the genus Oligonychus are separated into 2 clades (clades 1 and 19), with the Tetranychus species included in clade 19 (Figures 4B, 4D, 5B and 5D). Schizotetranychus species, with the exception of S. cercidiphylli, are separated into 3 clades (clades 3, 4 and 9), with the Sasanychus and Yezonychus species included in clade 9 (Figures 4B and 5B). In the ML tree (Figures 4B–4C), S. cercidiphylli and Eotetranychus species, with the exception of Eotetranychus uchidai Ehara, are paraphyletic with respect to clade 10. E. uchidai forms a sister group with Panonychus, Sasanychus, Schizotetranychus and Yezonychus species (Figure 4B, clade 8). In the Bayesian tree (Figures 5B–5C), a well-supported clade consisting of S. cercidiphylli and Eotetranychus species, with the exception of E. uchidai (clade 10: Bayesian posterior probabilities (BPP) = 0.96) clustered with clade 8.

Figure 4. Maximum likelihood (ML) phylogenetic tree of the sub-family Tetranychinae based on the 18S and 28S rRNA genes using the GTR Gamma model.

Bootstrap values (>50%) based on 1,000 replications are indicated at nodes. Each operational taxonomic unit is indicated by the voucher specimen no. and scientific name. Black circles with numbers indicate the clade no. which corresponds with the article. The tree is divided into three sections: (A) The entire tree, (B) Tetranychini-1, (C) Tetranychini-1, Eurytetranychini and Tetranychini-2 and (D) Tetranychini-3.

Figure 5. Bayesian phylogenetic tree of the sub-family Tetranychinae based on the 18S and 28S rRNA genes using the GTR Gamma model.

Bayesian posterior probabilities (>0.50) are indicated at nodes. Each operational taxonomic unit is indicated by the voucher specimen no. and scientific name. Black circles with numbers indicate the clade no. which corresponds with the article. The tree is divided into three sections: (A) The entire tree, (B) Tetranychini-1, (C) Tetranychini-1, Eurytetranychini and Tetranychini-2 and (D) Tetranychini-3.

As was observed in the COI tree, the 18S and 28S trees also show the monophyly of P. citri and P. osmanthi which are closely related species (Figures 4B and 5B, clade 6). S. cercidiphylli forms a well-supported clade with four Eotetranychus species (E. asiaticus, Eotetranychus boreus Ehara, E. cornicola and Eotetranychus toyoshimai Ehara & Gotoh) in both ML and Bayesian trees (Figures 4C and 5C, clade 11: BP/BPP = 93/1.00). On the other hand, closely related Eotetranychus species (Eotetranychus pruni (Oudemans), Eotetranychus querci Reeves and Eotetranychus uncatus Garman), which have long, flagellate and undulate aedeagi [25], did not cluster together in either tree (Figures 4C and 5C).

Discussion

Only a few studies have examined the molecular phylogeny of the sub-family Tetranychinae, and they often used genes or regions that had limited discriminating ability. As observed in previous studies, our tree based on the COI gene did not resolve deep-level phylogeny because of the low bootstrap values for deep nodes of tree (Figure 2). Therefore, we used the 18S and 28S rRNA genes for phylogenetic analyses because of their better discriminating ability. Indeed, our phylogenetic tree of the 18S and 28S sequences revealed several well-supported clades, allowing us to consider the phylogenetic relationships among the sub-family Tetranychinae.

Our phylogenetic trees based on the 18S and 28S rRNA genes suggest that the tribes Bryobiini and Petrobiini of the sub-family Bryobiinae are both monophyletic, but the tribe Tetranychini is polyphyletic because the monophyletic clade of Eurytetranychini is placed inside Tetranychini (Figures 4A and 5A). At the generic level, 4 genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) are polyphyletic. The phylogenetic tree separates the Oligonychus species into two clades (Figures 4B, 4D, 5B and 5D, clades 1 and 19). That is, the two clades comprising the genus Oligonychus coincide with their morphology based on the direction of curvature of the aedeagus. These results are in agreement with our COI phylogeny (Figure 2) and previous phylogenies based on the COI gene and ITS2 region [10], [12], [13], [14]. Although phylogenies based on the COI gene and ITS2 region could not establish the exact phylogenetic positions of the two clades of Oligonychus, our tree suggests that species whose aedeagi curve ventrally form a sister group with some of the Schizotetranychus species (Figures 4B and 5B, clade 2) and species whose aedeagi curve dorsally are more closely related to Tetranychus species whose aedeagi also curve dorsally (Figures 4D and 5D, clade 19). Though Oligonychus and Tetranychus can be distinguished by their empodium shape, our phylogenetic trees reveal that the shape of the aedeagi can help to discriminate these two genera.

Species of the genus Schizotetranychus and Eotetranychus appear to be polyphyletic within clade 12 (Figures 4B–4C and 5B–5C). Puzzlingly, S. cercidiphylli and E. uchidai are separated from other congeneric species in the tree. The placement of Eotetranychus species is different between the ML and Bayesian trees. In the ML tree (Figures 4B–4C), we could not establish the exact phylogenetic position of the species of Eotetranychus which are paraphyletic respect to clade 10 because bootstrap values are relatively low. On the other hand, in the Bayesian tree (Figure 5C), S. cercidiphylli and the Eotetranychus species, with the exception of E. uchidai, clustered into a well-supported clade (clade 10: BPP = 0.96). Similarly, the phylogenetic position of the genus Stigmaeopsis is resolved in the Bayesian analysis but not in the ML analysis. In the ML tree (Figure 4C), Stigmaeopsis species (clade 17) clustered with clade 13, which includes the Eurytetranychini species and some of the Tetranychini species, but the topology is not well supported (clade 16: BP = 50). In the Bayesian tree (Figure 5C), Stigmaeopsis species (clade 17) clustered with clade 13 with high Bayesian posterior probabilities (clade 16: BPP = 0.91). Although our data suggests that the Bayesian tree (Figures 5A–5D) is better supported than the ML tree (Figures 4A–4D), it is common knowledge that posterior probabilities are generally higher than bootstrap values [26].

Phylogenetic trees can be used to assess associations between spider mites and their host plants [13]. In the ML and Bayesian trees (Figures 4D and 5D), Oligonychus and Tetranychus species inhabiting gramineous plants (O. orthius, O. modestus, O. rubicundus and T. bambusae) clustered separately from other species and formed a monophyletic clade (Figures 4D and 5D, clade 18). Clade 4 includes Schizotetranychus brevisetosus Ehara, Schizotetranychus gilvus Ehara & Ohashi and Schizotetranychus shii (Ehara) which inhabit fagaceous plants (Figures 4B and 5B). Clade 9 include species irrespective of genus, which inhabit bamboo plants, Sasanychus akitanus (Ehara), Sasanychus pusillus Ehara & Gotoh, S. bambusae, S. recki and Yezonychus sapporoensis Ehara (Figures 4B and 5B). All Stigmaeopsis species inhabiting gramineous plants are separated from other Tetranychini species and appear to be monophyletic (Figures 4C and 5C, clade 17). These results indicate that the phylogenetic relationships of some species of spider mites are closely linked with their host plant, as reported in other phytophagous arthropods [27], [28], [29].

We consider the phylogenies of the Tetranychinae based on the 18S and 28S rRNA genes to be a major improvement over previous phylogenies because they reveal several well-supported clades that were not distinguished by phylogenetic relationships based on the COI gene and ITS2 region. Our finding that the tribe Tetranychini and four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) are polyphyletic indicates that the diagnostic morphological characters of tribes and genera of Tetranychinae need to be reconsidered. Although we examined a large number of species in this study, most of them were collected in Japan. Analyzing a number of undescribed genera remaining throughout the world may help achieve a deeper understanding of the phylogenetic relationships among the family Tetranychinae. In addition, a large number of nuclear genes need to be examined to resolve poorly understood relationships in the ML tree (Figures 4A–4D), such as the phylogenetic positions of the genera Eotetranychus and Stigmaeopsis.

Materials and Methods

Mites

Eighty-four strains representing 12 genera and two tribes in Tetranychinae, were used in this study and four strains of the tribes Bryobiini and Petrobiini of the sub-family Bryobiinae (Acari: Tetranychidae) were used as outgroups (Table 1). Mite samples that could be reared in the laboratory were maintained on leaf discs of common bean leaves (Phaseolus vulgaris L.), mulberry leaves (Morus bombycis Koidz.) or the original host plants placed on a water-saturated polyurethane mat in a plastic dish (90 mm diameter, 20 mm depth) at 25°C under a 16L-8D photoperiod until analysis. Samples that could not be maintained in the laboratory and samples that were imported from abroad were preserved in 99.5% ethanol for molecular analyses and 70% ethanol for morphological identification. Specimens were mounted in Hoyer’s medium and identified under phase-contrast and differential interference-contrast microscopes. Voucher specimens are preserved at the Laboratory of Applied Entomology and Zoology, Faculty of Agriculture, Ibaraki University under the serial voucher specimen numbers (Table 1).

DNA extraction, amplification, cloning and sequencing

Total DNA was extracted from the whole body of each female individual by using a Wizard Genomic DNA Purification Kit (Promega). Live female individuals for DNA samples and female individuals for voucher specimen were obtained from the same leaf discs. A few of the strains could not be maintained in the laboratory. For these strains, DNA samples were obtained from ethanol-preserved female individuals. The PCR primers are given in Table 2. The mitochondrial COI fragments were amplified using primer sets C1-J-1718 [30] and COI REVA [8] for species of 12 genera (Bryobia, Petrobia, Eurytetranychoides, Aponychus, Panonychus, Sasanychus, Schizotetranychus, Yezonychus, Eotetranychus, Oligonychus, Amphitetranychus and Tetranychus) and primer sets C1-J-1718-stig and COI REVA-stig for species of the genus Stigmaeopsis. COI sequences for Oligonychus and Tetranychus species were obtained from previously published data [10], [11]. PCR amplification was performed with the following profile: 3 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 45°C for COI, 60°C for 28S and 65°C for 18S and 1.5 min at 72°C. An additional 10 min at 72°C was allowed for last strand elongation. The resultant DNA solutions were purified by using MinElute PCR Purification Kit (Qiagen) and sequenced directly. Sequencing was carried out using the sequencing primers (Table 2) with a BigDye Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems) and on an ABI 3130×l automated sequencer.

Table 2. Primers used in polymerase chain reaction amplification and sequencing of the mitochondrial COI gene and the 18S and 28S rRNA genes.

Primer name			Sequence	Application	References
COI
	C1-J-1718	Forward primer	5′-GGAGGATTTGGAAATTGATTAGTTCC-3′	PCR amplification & sequencing	Simon et al. [30]
	COI REVA	Reverse primer	5′-GATAAAACGTAATGAAAATGAGCTAC-3′	PCR amplification & sequencing	Gotoh et al. [8]
	C1-J-1718-stig	Forward primer	5′-GGAGGTTTTGGTAATTGGTTAATCCC-3′	PCR amplification & sequencing	This study
	COI REVA-stig	Reverse primer	5′-GAAAGAACATAATGAAAATGAGCAAC-3′	PCR amplification & sequencing	This study
18S
	18S-1F	Forward primer	5′-ACCGCGAATGGCTCATTAAATCAGTT-3′	PCR amplification & sequencing	This study
	18S-2F	Forward primer	5′-TGGCCTCTGAGCCGACGATGTAT-3′	Sequencing	This study
	18S-2R	Reverse primer	5′-ACCCCATAGGTTCGACTGAAATC-3′	Sequencing	This study
	18S-5R	Reverse primer	5′-TCCAATAGATCCTCGTTAAAGGAT-3′	Sequencing	This study
	18S-8R	Reverse primer	5′-TCTCGTTCGTTATCGGAATTAAC-3′	Sequencing	This study
	18S-9F	Forward primer	5′-AGCTTCCGGGAAACCAAAGTTT-3′	Sequencing	This study
	18S-9R	Reverse primer	5′-AGGGCATCACAGACCTGTTATT-3′	Sequencing	This study
	18S-10F	Forward primer	5′-AGTTGGTGGAGTGATTTGTCTGGT-3′	Sequencing	This study
	18S-10R	Reverse primer	5′-ACAAAGGGCAGGGACGTAATCAA-3′	PCR amplification & sequencing	This study
28S
	28v-5′	Forward primer	5′-AAGGTAGCCAAATGCCTCATC-3′	PCR amplification & sequencing	Hillis and Dixon [31], Palumbi [32]
	28jj-3′	Reverse primer	5′-AGTAGGGTAAAACTAACCT-3′	PCR amplification & sequencing	Hillis and Dixon [31], Palumbi [32]

Data analysis

All sequences obtained were deposited in DDBJ/EMBL/GenBank International Nucleotide Sequence Databases under the accession numbers AB981203 to AB981240, AB926227 to AB926314 and AB926318 to AB926405 (Table 1). Sequences were aligned using the 'auto' option of the MAFFT software [33]. Gaps (insertions and deletions) included in the 18S and 28S rRNA sequences were treated using the 'automated1' option of the trimAl software [34], which trimmed ambiguous sites by using a heuristic selection of the automatic method based on similarity statistics. The homogeneity of nucleotide composition was checked using chi-square tests implemented in PAUP* version 4.0b10 software [35].

Maximum likelihood (ML) and Bayesian phylogenetic trees were constructed with RAxML [36] and MrBayes5D [37], respectively. We used the tribes Bryobiini and Petrobiini of the sub-family Bryobiinae as outgroups to root the tree. For all analyses, we used the GTR Gamma model selected by the Akaike Information Criterion (AIC) using the program Kakusan4 [38]. The RAxML search was executed for the best-scoring ML tree in one single program run (the ‘-f a' option) instead of the default maximum parsimony-starting tree. Statistical support was evaluated with 1,000 rapid bootstrap inferences. The MrBayes5D analyses were implemented with two parallel runs of 10 million generations each and using one cold and two incrementally heated Markov chains and sampling every 100 steps. Tracer v.1.6 [39] was used to assess if the search had reached stationarity and to check whether the sample sizes for each parameter (ESS>100) were adequate. The first 10% of the trees were discarded as burn-in and the consensus tree with Bayesian posterior probabilities was constructed based on the trees sampled after the burn-in.

Supporting Information

File S1

Aligned COI sequences in FASTA format.

(ZIP)

File S2

Aligned 18S sequences in FASTA format.

(ZIP)

File S3

Aligned 28S sequences in FASTA format.

(ZIP)

File S4

Aligned 18S sequences after deleting the ambiguous parts in FASTA format.

(ZIP)

File S5

Aligned 28S sequences after deleting the ambiguous parts in FASTA format.

(ZIP)

Data Availability

The authors confirm that, for approved reasons, some access restrictions apply to the data underlying the findings. All sequences obtained were deposited in DDBJ/EMBL/GenBank International Nucleotide Sequence Databases under the accession numbers AB981203 to AB981240, AB926227 to AB926314 and AB926318 to AB926405.

Funding Statement

Funding provided by Grant number 25292033, Japan Society for the Promotion of Science, http://www.jsps.go.jp/english/index.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.