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Zootaxa 3664 (4): 571–585 www.mapress.com / zootaxa / Copyright © 2013 Magnolia Press
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http://dx.doi.org/10.11646/zootaxa.3664.4.9 http://zoobank.org/urn:lsid:zoobank.org:pub:8F3BC3F8-AE22-4570-92CC-5F4750090C5C
Madeira’s ptyctimous mites (Acari, Oribatida) WOJCIECH NIEDBAŁA1,3 & MIROSLAWA DABERT2 Department of Animal Taxonomy and Ecology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland. E-mail:
[email protected] 2 Molecular Biology Techniques Laboratory, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland. E-mail:
[email protected] 3 Corresponding author 1
Abstract In the material recently collected in Madeira, 16 species of ptyctimous mites have been found. A new species of Austrophthiracarus rabacalensis Niedbała sp. nov. has been described. The presence of P. globosus and S. (R.) ortizi, reported earlier from Madeira has not been confirmed, but P. anonymus and P. montanus, so far not reported from this island, have been found. All 16 species identified in the material from Madeira studied occur in the Palaearctic Region; four of them are endemites, seven occur in western Palaearctic, four are panpalaearctic, while one is a semicosmopolitan. Morphological analysis has revealed a high similarity of two endemic species of Madeira with two European species: Steganacarus (Steganacarus) crassisetosus is similar to Steganacarus (Steganacarus) applicatus, while Steganacarus (Steganacarus) similis to Steganacarus (Steganacarus) spinosus. DNA-barcode analysis using COI and D2 28S rDNA sequences confirmed the species status of these four species. The phylogenetic analyses of COI amino acid data and D2 28S rDNA sequences suggest a closer relationship between S. (S.) crassisetosus and S. (S.) applicatus, pointing to a great genetic distance between S. (S.) spinosus and the other species of Steganacarus (Steganacarus). Key words: Steganacaridae, DNA barcoding, COI, D2 28S rDNA, molecular phylogeny, morphological analysis, new species
Introduction The Madeira Archipelago and the other islands from Macaronesian Archipelago are of volcanic origin so their flora and fauna are a result of passive dispersion. The most probable colonisation route was from south-western Iberia and from Morocco (Bernini and Magari 1993). The information on the oribatid mite fauna from Madeira are skimpy and have been presented in only a few papers. Willmann (1939) has proved the presence of 6 species and 1 subspecies of ptyctimous mites, and 2 of these species and 1 subspecies have been described as new to science. The types of these new species are not preserved (Bernini & Avanzati 1989). Bernini and Magari (1993) redescribed the species Steganacarus similis described by Willmann and reported the presence of one more species Steganacarus (Rhacaplacarus) ortizi. Pérez-Iñigo (1988) and Niedbała (2011, 2102) in joint works have reported the presence of all species known from Madeira. The aim of this study is the identification of the ptyctimous mite species from recently obtained material from Madeira, present the current status of oribatid fauna there, and describe a new species. Moreover, on the basis of morphological and molecular data, the diagnoses of the other ptyctimous species described by Willmann (1939) are given together with a taxonomic comment.
Material and methods Animal material. Material studied in this work comes from four soil samples collected in February 2012. In these samples the following species of ptyctimous mites were identified: Accepted by H. Schatz: 8 Apr. 2013; published: 28 May 2013
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Madeira, Western Part, Paul da Serra plateau, 1200 m.a.s.l., under Erica, 02.02. 2012, leg. W. Niedbała Phthiracarus anonymus Grandjean, 1933 – 6 Phthiracarus montanus Pérez-Íñigo, 1969 – 4 Madeira, Western Part, near Rabaçal, litter under Erica and Laura trees, 04.02. 2012, leg W. Niedbała Acrotritia duplicata (Grandjean, 1953) – 3 Phthiracarus anonymus – 7 Phthiracarus membranifer Parry, 1979 – 2 Phthiracarus nitens (Nicolet, 1855) – 6 Steganacarus (Steganacarus) crassisetosus (Willmann, 1939) – 11 Steganacarus (Steganacarus) similis (Willmann, 1939) – 45 Austrophthiracarus parainusitatus sp. nov.– 1 Atropacarus (Atropacarus) plakatisi (Mahunka, 1979) – 3 Madeira, Funchal, tropical jardin, litter under trees, 02.02. 2012, leg. W. Niedbała Phthiracarus anonymus – 2 Phthiracarus membranifer – 2 Phthiracarus nitens – 2 Steganacarus (Steganacarus) magnus (Nicolet, 1855) – 1 Madeira, Eastern Part, near Balcǒes, litter from “Laurisilva” forest, 05.02. 2012, leg. W. Niedbała Phthiracarus anonymus – 2 Phthiracarus membranifer – 44 Phthiracarus montanus – 1 (Fig. 1A–C) Steganacarus (Steganacarus) crassisetosus – 11 Steganacarus (Steganacarus) similis – 28 Atropacarus (Atropacarus) plakatisi – 3 Preparatory methods for morphological examination and terminology are based on those of Niedbała (2011). Molecular methods. Total genomic DNA was extracted from individual specimens using a non-destructive method as described by Dabert J. et al. (2008). A 670-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with primers bcdF01 (5'-CATTTTCHACTAAYCATAARGATATTGG-3') and bcdR04 (5'-TATAAACYTCDGGATGNCCAAAAAA-3') (Dabert M. et al. 2010), and an 850-bp fragment of the nuclear 28S rDNA, including the hypervariable D2 region, was amplified with primers 28SF0001 (5'ACCCVCYNAATTTAAGCATAT-3') and 28SR0990 (5'-CCTTGGTCCGTGTTTCAAGAC-3') (Mironov et al. 2012). PCRs were carried out in 5 µl reaction volumes containing 2.5 µl Type-it Microsatellite PCR Kit (Qiagen, Hilden, Germany), 0.25 µM of each primer, and 1 µl of DNA template using a thermocycling profile of one cycle of 5 min at 95 °C followed by 35 steps of 30 sec at 95 °C, 90 sec at 50 °C, 1 min at 72 °C, with a final step of 5 min at 72 °C. After amplification, the PCR were diluted with 10 µl of water and 5 µl of the diluted PCR reaction were analysed by electrophoresis on a 1% agarose gel. Samples containing visible bands were directly sequenced in the forward direction by using 1 µl of the PCR reaction and 50 pmoles of sequencing primer. Sequencing was performed with a BigDye Terminator v3.1 on an ABI Prism 3130XL Analyser (Applied Biosystems). Sequence chromatograms were checked for accuracy using FinchTV 1.3.1 (Geospiza Inc.). The nucleotide sequences were aligned manually in GeneDoc v. 2.7.000 (Nicholas & Nicholas 1997). DNA sequences for COI were converted into amino acids and no amino acid insertion/deletion was found. Nucleotide frequencies, neighbour-joining (NJ) analysis, and pairwise distance calculations between sequences were performed in MEGA 5.01 (Tamura et al. 2011); the distances were computed using the Kimura two parameter (K2P) distance model (Kimura 1980) for all sequence positions. Contigs of the 28S rDNA sequences were aligned and assembled manually in GenDoc and the distances between sequences were computed using the K2P distance model with MEGA 5.01. All positions containing insertion/deletion were eliminated only in pairwise sequence comparisons (pairwise deletion option).
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The COI phylogenetic tree was reconstructed using amino acid sequence data in TREEFINDER (Jobb 2011) by maximum likelihood (ML) method using mtArt model of amino acid replacement (Abascal et al. 2007) with G = 0.3105586. The confidence of inferred relationships was assessed using two methods: the bootstrap analysis (BS) (500 replicates) and the local rearrangements of expected-likelihood weights (LR-ELW edge support; 1000 replicates) (Strimmer & Rambaut 2002) as implemented in TREEFINDER. Likelihood scores for 28S rDNA sequences were computed with PHYML (Guindon & Gascuel 2003). Nucleotide substitution model selected by jModeltest 0.1.1 (Posada 2008) using Akaike Information Criterion (AIC) was TPM3uf + G with gamma shape = 0.0160. The D2 ML tree was reconstructed using Garli (Zwickl 2006) with 100 search replications and support was calculated by 500 bootstrap replications. The D2 NJ tree was reconstructed using MEGA 5.01 with 1000 bootstrap replications. Sequence data used for molecular analysis are listed in Table 1. All sequences are deposited in GenBank under accession numbers indicated in Table 1. The alignments are available upon request. TABLE 1. Sequence data used in molecular analysis. Species
Sample code
Localization
Steganacarus (S.) similis S. (S.) similis S. (S.) similis S. (S.) similis S. (S.) similis S. (S.) applicatus S. (S.) applicatus S. (S.) applicatus S. (S.) applicatus S. (S.) applicatus S. (S.) spinosus S. (S.) spinosus S. (S.) spinosus S. (S.) spinosus S. (S.) spinosus S. (S.) spinosus S. (S.) crassisetosus S. (S.) crassisetosus S. (S.) crassisetosus S. (S.) crassisetosus S. (S.) crassisetosus S. (S.) crassisetosus S. (S.) magnus S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi
AA527 AA528 AA529 AA530 AA532 AA535 AA537 AA539 AA540 AA542 AA545 AA546 AA547 AA548 AA549 AA552 AA553 AA554 AA555 AA556 AA557 AA560 Env004
Madeira Madeira Madeira Madeira Madeira Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Madeira Madeira Madeira Madeira Madeira Madeira Poland La Gomera La Gomera La Gomera La Gomera La Gomera La Gomera La Gomera La Gomera La Gomera Tenerife Tenerife Tenerife Tenerife Tenerife
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GenBank Acc. nos. COI 28S JX891539 JX891564 JX891540 JX891541 JX891542 JX891543 JX891544 JX891565 JX891545 JX891546 JX891547 JX891548 JX891549 JX891562 JX891550 JX891551 JX891552 JX891553 JX891554 JX891555 JX891563 JX891556 JX891557 JX891558 JX891559 JX891560 JF264105* JX891566 AJ414177* AJ414189* AJ414175* AJ414188* AJ414192* AJ414190* AJ414191* AJ414176* AJ414178* AJ414179* AJ414180* AJ414196* AJ414181* AJ414193* ......continued on the next page
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TABLE 1. (continued) Species S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi S. (S.) carlosi Steganacarus sp. S. (S.) carusoi S. (S.) guanarteme S. (S.) tenerifensis S. (S.) tenerifensis S. (S.) tenerifensis Phthiracarus longulus
Sample code
Env222
Localization Tenerife Tenerife Tenerife Gran Canaria Gran Canaria Gran Canaria Gran Canaria La Gomera Sardinia Gran Canaria Tenerife Tenerife Tenerife Poland
GenBank Acc. nos. COI AJ414194* AJ414182* AJ414195* AJ414199* AJ414200* AJ414185* AJ414201* AJ414186* AJ414187* AJ414184* AJ414197* AJ414183* AJ414198* GQ864385*
28S JX891561
*sequences drawn from GenBank database.
Results Description of one new species Austrophthiracarus rabacalensis Niedbała sp. nov. (Fig. 1D–J) Measurements of holotype. Prodorsum: length 338, width 227, height 146; setae of prodorsum: sensillum 126, interlamellar 101, lamellar 83, rostral 76, exobothridial 30; notogaster: length 616, width 394, height 434; setae of notogaster: c1 266, c3 38, cp 81, h1 341, p1 316, h3 40, p4 35; genitoaggenital plate: 156 × 101, anoadanal plate: 242 × 106. Integument. Colour light brown. Body shagreen, strongly sculptured. Surface of body without concavities. Prodorsum. Sigillar fields long and narrow. Lateral carinae short and feeble, reaching the sinus. Sensilla long, slightly sickle-shaped, of equal width, without head. Setae of medium size, attenuate. Notogaster carries 16 pairs of rigid setae, majority of them very long (c1>c1–d1), flagellate. Only setae c3, cp, h3, p4 are short needleform, additional setae situated between h1–p1 (h1’), the distance between setae p3 and p4 is large, setae c1 and c3 near anterior margin, setae c2 slightly removed from the margin; vestigial setae f1 anteriorly of setae h1; all four pairs of lyrifissures ia, im, ip and ips present. Ventral region. Seta h of mentum longer than distance between them. Formula of genital setae: 4+2:3, setae g7longer than g6, all setae g6-9 longer than setae g1-5. Anoadanal plates with long, flagellate setae, adanal setae ad1 and 9 ad2 longer than other setae. Legs. Chaetome of legs of “complete type”. Setae d of femora I located at distal end of article, setae v” longer than half of length of v’ setae. Material examined. Holotype. Madeira, Western Part, near Rabaçal, litter under Erica and Laura trees, 04.02. 2012, leg W. Niedbała. Type deposition. Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Poznan. Comparison. The most similar species Austrophthiracarus parainusitatus Niedbała & Stary, 2011 from Azores is distinguishable from the new species by the absence of heterotrichy of notogastral setae. The second similar species is Austrophthiracarus inusitatus (Niedbała, 1983) from Far East of Asia by the shape of sensilla, shape and length of prodorsal setae, length and arrangement of genital, anal and adanal setae. It is distinguishable from the new species by the similar shape and length of notogastral setae and presence of two pairs of lyrifissures. Etymology. The specific name refers to the valley of Rabaçal, Madeira.
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FIGURE 1 A-J. A–C. Phthiracarus montanus Pérez-Íñigo, 1969: A, prodorsum, lateral view; B, anterior part of notogaster, lateral view; C, trochanter and femur I. D-J. Austrophthiracarus rabacalensis Niedbała sp. nov. D, prodorsum, dorsal view; E, prodorsum, lateral view; F, mentum of subcapitulum; G, genitoaggenital plate; H, anoadanal plate, I, lateral view of opisthosoma, J, trochanter and femur I.
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Diagnoses of two known species. Two known Steganacarus species, Steganacarus (Steganacarus) crassisetosus and Steganacarus (Steganacarus) similis are morphologically very similar to the palaearctic species S. (S.) applicatus and S. (S.) spinosus, respectively. Both S. (S.) applicatus and S. (S.) spinosus are the species of western palaearctic origin, and S. (S.) applicatus has been also found in Algeria.
Steganacarus (Steganacarus) crassisetosus (Willmann, 1939) (Figs. 2A–G) Steganacarus applicatum (Sell.) var. crassisetosus n. var. Willmann, 1939
Measurements of specimen from Balcoes, Madeira. Prodorsum: length 379, height 192, width 288; setae of prodorsum: sensillum 101, interlamellar 177, lamellar 126, rostral 76, exobothridial 30; notogaster: length 818, height 566, width 555, setae of notogaster: c1 185, c1/c1–d1=1.0, h1 106, p1 91; genitoaggenital plates: 227 × 192, anaoadanal plates: 268 × 172. Diagnosis. Large species. Colour brown. Integumental body sculpture slightly areolate. Prodorsum with distinct median crista; median and lateral fields very long; lateral carinae absent; sensilla long, thick covered with spines, sickle-shaped; interlamellar and lamellar setae resemble gastronotic setae covered with small spines, rostral setae long, rough, curved sagittally, in>le>ro>ex. Notogaster with flattened setae covered with spines, setae h1 and p1 located perpendicularly to the surface of notogaster, the remaining setae procumbent; setae h1 and p1 considerably thicker and shorter, about the half of length of procumbent setae; setae c1=c1–d1; setae c3 near the anterior margin, c1 further away and c2 in the most distant position; vestigial setae f1 posteriorly of setae h1; two pairs of lyrifissures ia and im. Ventral region: h>h–h; genitoaggenital plates with setal formula: 6(4+2): 3; anoadanal plate with four setae at proximal margin which diminish towards the anterior margin, setae ad3 very short, spiniform, an/not=0.3. Chaetome of legs complete, setad d on femora I slightly remote from anterior margin. Comparison. Willmann (1939) has particularly emphasised one character differentiating crassisetosus from the main form applicatus (Fig. 2H–M), that is short, thick and perpendicular setae h1 and p1 much shorter than the others; in applicatus the length of these setae is similar to that of the other ones. Moreover, Willmann claims that the size of crassisetosus is larger, however, in view of great variation in the size of phthiracaroid mites individuals, this feature should be treated with caution. There are two main morphological features differing S. (S.) applicatus and S. (S.) crassisetosus; the perpendicular setae h1 and p1 in S. (S.) crassisetosus are thick and shorter (h1: e1 = 0.55) than procumbent setae, while in S. (S.) applicatus, the setae h1 and p1 are of the same thickness and similar length (h1, e1 = 0.85) as procumbent setae. Rostral setae in S. (S.) crassisetosus are long, rough, curved sagittally but rostral setae in S. (S.) applicatus are shorter, spiniform and diriged forward. Steganacarus (S.) crassisetosus is also similar to S. (S.) carusoi (Bernini & Avanzati, 1989) (Western Palaearctic species, reported from the Maghrebian and Atlanto-Mediterranean region). These species have perpendicular setae h1 and p1 and rostral setae of similar length directed inward but S. (S.) carusoi has shorter lamellar setae, perpendicular setae h1 and p1 of the same length as the other procumbent setae, setae c1 of notogaster situated slightly nearer to anterior border than c3 setae and setae ad3 in distal half are rugose or covered with small spines (versus shorter perpendicular setae than procumbent setae, greater distance of c1 setae from anterior border and small, spiniform ad3 setae in S. (S.) crassisetosus).
Steganacarus (Steganacarus) similis Willmann, 1939 (Fig. 3A–G) Steganacarus similis: Bernini and Magari 1993
Measurements of specimen from Rabaçal, Madeira. Prodorsum: length 257, height 111, width 202; setae of prodorsum: sensillum 96, interlamellar 139, lamellar 101, rostral 45, exobothridial 20; notogaster: length 596, height 353, width 364, setae of notogaster: c1 88, c1/c1–d1=0.7, h1 and p1 101; genitoaggenital plates: 156 × 106, anaoadanal plates: 207 × 116.
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FIGURE 2 A–M. A–G. Steganacarus (Steganacarus) crassisetosus (Willmann, 1939): A, prodorsum, dorsal view; B, prodorsum, lateral view; C, mentum of subcapitulum; D, genitoaggenital plate; E, anal and adanal setae; F, lateral view of opisthosoma, G, trochanter and femur I. H–M. Steganacarus (Steganacarus) applicatus (Sellnick, 1920) (specimen from Poland): H, prodorsum, dorsal view; I, prodorsum, lateral view; J, genitoaggenital plate; K, anoadanal plate; L, femur I; M, lateral view of opisthosoma.
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FIGURE 3 A–N. A–G. Steganacarus (Steganacarus) similis (Willmann, 1939): A, prodorsum, dorsal view; B, prodorsum, lateral view; C, mentum of subcapitulum; D, genitoaggenital plate; E, anoadanal plate; F, lateral view of opisthosoma; G, trochanter and femur I. H–N. Steganacarus (Steganacarus) spinosus (Sellnick, 1920) (specimen from Poland): H, prodorsum, dorsal view; I, prodorsum, lateral view; J, mentum of subcapitulum; K, genitoaggenital plate; L, anoadanal plate; M, lateral view of opisthosoma; N, trochanter and femur I.
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Diagnosis. Surface covered with feeble concavities. Colour brown. Prodorsum: median field narrow and longer than the laterals; lateral carinae absent; sensilla long, narrow, sickle-shaped covered with spines; setae, other than the exobothridial covered with distinct spines in distal half, rostral setae directed inward. Notogaster with rigid, short (c1h–h; formula of genital setae: 6(4+2): 3, anoadanal plate with fairly long setae, ad1 and ad2 are longest, an/not=0.3. Chaetome of legs reduced; setae s on tarsi I are missing; v”/v’=4.4. Comparison. This species is very similar to the Paneuropean species S. (S.) spinosus (Fig. 3H–N). The comparison was made with the specimens from the population collected in Poland (Sudety, Wielka Sowa). Measurements of specimen of S. (S.) spinosus (Sellnick, 1920) from Wielka Sowa, Poland. Prodorsum: length 303, height 126, width 212; setae of prodorsum: sensillum 94, interlamellar 152, lamellar 63, rostral 43, exobothridial 40; notogaster: length 606, height 404, width 434, setae of notogaster: c1 121, c1/c1–d1=0.66, h1 and p1 101; genitoaggenital plates: 167 × 126, anaoadanal plates: 202 × 131. Steganacarus (S.) similis differs from S. (S.) spinosus also by two main morphological features. S. (S.) similis from Madeira has longer lamellar setae (le/in > 0.7), S. (S.) spinosus has much shorter lamellar setae (le/in < 0.5); rostral setae in S. (S.) similis are diriged inward but rostral setae of S. (S.) spinosus are diriged forward. Willmann (1939) differentiated S. (S.) similis from S. (S.) spinosus – see also Willmann (1931), but in the comparison he has put too much emphasis on the body size and he was incorrect in claiming that the setae of S. (S.) spinosus are smooth and pointed distally. Bernini and Magari (1993) have determined the neotype of S. (S.) similis. They have pointed to some small morphological differences between S. (S.) similis with S. (S.) spinosus: in the shape of sensilla, shape of anoadanal setae, lack of setae a on the first tarsi, the length of anoadanal setae decreases gradually from ad1 to an2 in S. (S.) similis but in S. (S.) spinosus setae ad1 and ad2 are longer than setae an1 and an2. In the material for comparison performed in this study, i.e. from the S. (S.) similis population from Madeira and S. (S.) spinosus from Poland, these differences were not confirmed; the shapes of sensilla and anoadanal setae are similar in both species and also setae ad1 and ad2 are the longest. In view of a considerable morphological similarity of these two species, an attempt was made to establish their genetic relationship.
Molecular analysis of the species status of S. (S.) crassisetosus, S. (S.) similis, S. (S.) spinosus, and S. (S.) applicatus The COI alignment for the distance calculations comprised 620 bp of unambiguous sequence data for 22 specimens of four Steganacarus species: S. (S.) applicatus (5), S. (S.) crassisetosus (6), S. (S.) similis (5), and S. (S.) spinosus (6). The number of variable sites was 207 and the estimated base frequencies were as follows: T = 0.4419, C = 0.1750, A = 0.2196, G = 0.1635. The average transition to transversion ratio (R) = 1.29 for all variable sites. NJ analysis of the COI sequences revealed four well-supported clades corresponding to the tested species (not shown). Estimates of average evolutionary divergence (K2P) were about one percent within clades grouping: S. (S.) applicatus (1.02%, SE 0.28), S. (S.) spinosus (1.21%, SE 0.27), and S. (S.) similis (1.26%, SE 0.34). Nucleotide substitutions observed in the individual clades were synonymous and did not change the amino acid sequence. The highest divergence was found in S. (S.) crassisetosus clade (3.78%, SE 0.55) but even in this case all nucleotide substitutions were synonymous. The average genetic distance among reconstructed clades was 23.03% (SE 2.15) and ranged from 20.14% (SE 1.94) between S. (S.) crassisetosus and S. (S.) applicatus to 27.17% (SE 2.47) between S. (S.) spinosus and S. (S.) similis. This level of genetic distance outnumbered intraspecific divergence and strongly support the species status of the analysed taxa. The COI nucleotide sequences were published in GenBank as DNA barcodes for S. (S.) similis (Acc. JX891540-43), S. (S.) applicatus (Acc. JX891544-48), S. (S.) spinosus (Acc. JX891549-54), and S. (S.) crassisetosus (Acc. JX891555-60). The analysis of the nuclear D2 marker corroborated the COI results. More than five specimens from each species have been sequenced in D2 region of 28S rDNA and no intraspecific polymorphism was found among sequences from individuals belonging to the same species. The final D2 28S rDNA alignment for the distance calculations comprised 373 nucleotide sites in which 34 were variable. The mean base frequencies were: A =
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0.2347, C = 0.2043, G = 0.2737, T = 0.2872, and transition to transversion ratio (R) was 2.35. Estimates of average genetic distance over sequence pairs representing each species showed that S. (S.) spinosus is the most diverged from the three other species (8.37%, SE 1.50%), while the average divergence among S. (S.) crassisetosus, S. (S.) similis, and S. (S.) applicatus was 2.03% (SE 0.73). The genetic distances between S. (S.) crassisetosus and S. (S.) applicatus as well as between S. (S.) spinosus and S. (S.) similis did not prove unambiguously their relationship. To find out the evolutionary relations among them, a phylogenetic analysis was made on the basis of greater representation of the species belonging to Steganacaridae.
FIGURE 4 Maximum likelihood COI amino acid tree of S. crassisetosus, S. similis, S. spinosus, S. applicatus, and other Steganacaridae. The support of branches is given as LR-ELW edge support for 1000 ML replications (left) and bootstrap 500 replicates (right). Only support above 50% (LR-ELW)/(BS) is shown.
Phylogenetic relationships among S. (S.) crassisetosus, S. (S.) similis, S. (S.) spinosus, S. (S.) applicatus, and other Steganacaridae The alignment for phylogenetic tree reconstruction using COI sequence data comprised of 129 amino acid positions for 51 sequences. Twenty two sequences from this study were compared with the other known COI sequences for Steganacaridae: several species inhabiting Canary Islands and Sardinia (Salomone et al. 2002), one representative of European species S. (S.) magnus (JF264105), and Phthiracarus longulus used as the outgroup (for details see Table 1). Maximum likelihood (ML) analysis based on COI amino acid data confidently recovered all analysed species (Fig. 4). Although most of the recovered evolutionary relationships among analysed Steganacaridae remain unresolved due to a low or no nodal support, the ML analysis of the COI sequences showed that S. (S.) spinosus is the most distant species from the others and that there is no support for a close relationship between S. (S.) applicatus and S. (S.) crassisetosus. The ML COI tree revealed S. (S.) spinosus basally to wellsupported (LR-ELW edge support =93, BS=83) clade containing the remainder of the taxa. In this group, European
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Steganacaridae were placed basally to island clades, however this placement was not supported by the tests. Canary Island species formed well-supported monophyletic clades corresponding to each island, with the exception of the position of Steganacarus sp. from La Gomera (GenBank Acc. no AJ414186) which grouped with two species from Madeira and one from Sardinia, but there was lack of support for this relationship. The COI amino acid sequences provided weak support (57/-) for close relationship of S. (S.) crassisetosus and S. (S.) similis. Both species were found in a clade with the S. (S.) carusoi, but the latter relationship remains unresolved due to the lack of support for S. (S.) carusoi + S. (S.) similis clade. The alignment for phylogenetic analysis using the sequence data from the D2 region of 28S rDNA comprised of 375 nucleotide positions for 6 species: S. (S.) spinosus, S. (S.) crassisetosus, S. (S.) similis, S. (S.) applicatus, S. (S.) magnus, and Phthiracarus longulus used as the outgroup. The number of variable sites was 62. Phylogenetic analysis of the D2 marker performed using both the ML and NJ methods confirmed a distant relationship of S. (S.) spinosus to the other analysed Steganacaride species. In general, the two methods generated trees with the same topology (Fig. 5) where S. (S.) spinosus was placed basally to well-supported (BS = NJ 91%, ML 56%) clade (S. (S.) magnus, (S. (S.) similis, (S. (S.) crassisetosus, S. (S.) applicatus)). Moreover, both analyses recovered with moderate support (NJ 67%, ML 65%) a closer relationship between S. (S.) applicatus and S. (S.) crassisetosus.
FIGURE 5. Maximum likelihood (ML) D2 28S rDNA tree of S. crassisetosus, S. similis, S. spinosus, and S. applicatus. Neighbour-joining (NJ) analysis produced a tree having the same topology. The support of branches is given as bootstrap 500 (ML) and 1000 (NJ) replicates. Only support above 50% (NJ)/(ML) is shown.
Discussion The current knowledge of ptyctimous species known from Madeira. The most important paper on the fauna of oribatid mites of Madeira is that by Willmann (1939). Besides the two above diagnosed species it would be worthwhile to comment on the other ptyctimous mites species found and described by this author. Phthiracarus globosus (C.L.Koch 1841) could have been mistaken for Phthiracarus nitens (Nicolet, 1855). When describing Phthiracarus ferrugineus (C.L. Koch), Willmann refers to the redescription made by Jacot (1936). However, the species redescribed by Jacot was proved to be Phthiracarus longulus (C.L. Koch, 1841) (Niedbała 2011). Phthiracarus longulus has not been found in Madeira as yet, although its presence is highly probable. I suppose that the Phthiracarus ferrugineus specimen found by Willmann (1939) can prove to be
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Phthiracarus anonymus Grandjean, 1933, to which it is morphologically similar and which has been reported from Madeira. The Phthiracarus lentulus (C.L.Koch, 1841) identified by Willmann most is probably Phthiracarus membranifer Parry, 1979. This supposition stems from the fact of finding in the samples from Madeira in 2012, a relatively large number of P. membranifer (Parry, 1979) individuals at the absence of P. lentulus (C.L.Koch, 1841). Willmann (1939) identified P. lentulus on the basis of redescription of Jacot (1936). However, Jacot did not take into regard the chaetome of legs in his descriptions of species. It is vital as the species P. membranifer and P. lentulus are very similar and the main feature distinguishing them is the "complete chaetotaxy" in P. lentulus and the "incomplete chaetotaxy" in P. membranifer (absence of setae v' on femora I and absence of setae l’ on genua IV). TABLE 2. The current list of ptyctimous species known from Madeira. Species
Range
Oribotritia berlesei (Michael, 1898): Niedbała (2011)
panpalaearctic
Austrotritia herenessica (Pérez-Íñigo,1986): Niedbała (2011)
western palaearctic, also in Gomera
Euphthiracarus monodactylus (Willmann, 1919): Niedbała (2011)
panpalaearctic
Acrotritia duplicata (Grandjean, 1953)
panpalaearctic
Phthiracarus anonymus Grandjean, 1933
semicosmopolitan, also in Algeria
? Phthiracarus ferrugineus: Willmann (1939) Phthiracarus membranifer Parry, 1979
panpalaearctic, also in Algeria
? Phthiracarus lentulus (C.L.Koch, 1841): Willmann (1939) Phthiracarus montanus Pérez-Íñigo, 1969
western palaearctic, also in Algeria
Phthiracarus nitens (Nicolet, 1855)
western palaearctic, also in Algeria and Tenerife
? Phthiracarus globosus (C. L. Koch, 1841): Willmann (1939) Phthiracarus torosus Willmann, 1939
endemic (enigma)
Steganacarus (Rhacaplacarus) ortizi Pérez-Íñigo, 1969: Bernini and Magari (1993)
western palaearctic
Steganacarus (Steganacarus) applicatus (Sellnick, 1920): Niedbała (2011, 2012)
western palaearctic, also in Algeria
Steganacarus (Steganacarus) crassisetosus Willmann, 1939
endemic
Steganacarus similis Willmann, 1939
endemic
Steganacarus (Steganacarus) magnus (Nicolet, 1855)
western palaearctic
Austrophthiracarus parainusitatus sp. nov.
endemic
Atropacarus (Atropacarus) plakatisi (Mahunka, 1979)
western palaearctic, also in Algeria and Morocco
“?”—the presence of species is questionable.
The validity of the Phthiracarus torosus described by Willmann (1939) on the basis of one single individual seems highly improbable. It is very similar to P. membranifer (size of sensilla, length and size of prodorsal and notogastral setae) but it has an anterior collar and a clear median ridge on notogaster, running along the whole dorsal side of notogaster. Also the arrangement of genital setae and the ratio of the lengths of setae on anoadanal plates seem unusual. In the material studied in this work the presence of P. globosus and S. (R.) ortizi, reported from Madeira (Willmann 1939; Bernini & Magari 1993) is not confirmed, but other species, so far not reported from this island, P. anonymus and P. montanus have been found. All 16 species identified in the material from Madeira studied occur in the Palaearctic Region. Four of them are endemites, seven occur in western palaearctic, four in the panpalaerctic region, while one is of semicosmopolitan distribution. Seven of the species occur in Maghreb countries, and two species in the Canary Islands. Analysis of the zoogeographic elements reveals mainly the European influence, but north African influence is also pronounced. The influence of continental fauna on that of Madeira could however have changed in the past. It
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should be remembered that Madeira had changed its relation to Africa in the last few million years (Bernini & Magari 1993). The current list of ptyctimous species known from Madeira is presented in Table 2. Sequence divergence in Steganacarus. Molecular analysis of the mitochondrial COI along with the hypervariable D2 region of 28S rDNA from nuclear DNA unambiguously confirmed the species status of S. (S.) spinosus, S. (S.) applicatus, S. (S.) crassisetosus, and S. (S.) similis. The average COI sequence divergences within steganacarid species were about one percent (K2P) which is at the level observed in other actinotrichid mites. For example, similar distances among COI sequences were found in two closely-related water-mite species Hygrobates nigromaculatus and H. setosus (Prostigmata, Parasitengona) (Martin et al. 2010), eriophyoid mites belonging to the complex Abacarus hystrix (Prostigmata, Eriophyoidea) (Skoracka & Dabert 2010), and congeneric feather mite species (Astigmata, Analgoidea), e.g. Glaucalges tytonis (Analgoidea, Xolalgidae) (Dabert J. et al. 2010) or Proctophyllodes valchukae (Analgoidea, Proctophyllodidae) (Mironov et al. 2012). The relatively high level of intraspecific genetic diversity in COI sequences from S. (S.) crassisetosus (3.78%) probably not indicate a cryptic speciation, because all the nucleotide substitutions were synonymous and there was no intraspecific sequence variability in the hypervariable region D2 of 28S rDNA. Moreover, a similar high intraspecific divergence was found in populations of Stegancarus carlosi inhabiting the Canary Islands (Salomone et al. 2002). This high divergence could be explained by genetic isolation of populations inhabiting the different islands and subsequent remixing events resulting from human activities. However, we did not observe this pattern in (S.) similis, the second island species analyzed in this study. Thus, in order to determine genetic variability and patterns of this variation within Steganacarus more detailed sampling and different molecular markers (e.g. microsatellites) are necessary. Relatively high interspecific distances among COI sequences from S. (S.) spinosus, S.(S.) applicatus, S. (S.) crassisetosus, and S. (S.) similis were at the same level as in other congeneric actinotrichid species. For example, the water mite sibling species pair, H. nigromaculatus and H. setosus, differs in COI sequences by ca. 20% (Martin et al. 2010). Similar distances were found among closely-related eriophyoid species (Skoracka & Dabert M. 2010), feather mites (Dabert J. et al. 2008), and other oribatids belonging to Scutoverticidae (Schäffer et al. 2008; Schäffer et al. 2010). To date, there is no information about intraspecific variability in the D2 28S rDNA region in oribatid mites. We observed no variation among D2 sequences within each species analysed in the present study. In other actinotrichid lineage, Prostigmata, this sequence region was noticed to be polymorphic within species (Martin et al. 2010; Skoracka & Dabert M. 2010), however, at a very low level (20% for COI) suggests that they are not closely-related sibling species. The ML analysis of COI amino acid data did not support evolutionary affinities of the four studied species with the other analysed Steganacaridae because of lack of support for recovered clades containing S. (S.) applicatus, S. (S.) crassisetosus, and S. (S.) similis. The D2 tree was reconstructed using fewer species since there is no sequence data of the D2 region of 28S rDNA for the Steganacaridae drawn from GenBank in COI analysis. Nevertheless, both NJ and ML analyses of D2 data reconstructed trees of the same topology and similar support for a very distant relationship of S. (S.) spinosus to the remaining species and the monophyly of S. (S.) applicatus + S. (S.) crassisetosus clade.
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The molecular phylogenetic analyses suggest closer relationship between S. (S.) crassisetosus and S. (S.) applicatus, pointing to a great genetic distance between S. (S.) spinosus and the other species of Steganacarus (Steganacarus). However, in order to solve the phylogenetic relationships within this group it would be necessary to carry out a more detailed study on a greater number of species representing Steganacaridae and some additional external groups as the results obtained in this study suggested that Phthiracarus longulus used as an outgroup made a polytomy with S. (S.) spinosus.
Acknowledgments The present study was supported by the Polish MSHE grant No N N303 017937.
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