Diabrotica wartensis Cabrera & Sosa-Gómez, 2008

Cabrera, Nora, Sosa, Daniel R. & Micheli, Adriana, 2008, Morphological and molecular characterization of a new species of Diabrotica (Coleoptera, Chrysomelidae, Galerucinae), Zootaxa 1922, pp. 33-46 : 35-41

Diabrotica wartensis Cabrera & Sosa-Gómez , New species

( Figs. 1 –24)

Diagnosis. Small-sized species, body oval, slightly convex. Coloration bright green. Genal space one-fourth or a little more of the maximum length of the eye. Antennomere 3 little longer than 2 in males and subequal in females, together more than one half the length of 4. Elytra with a single binding patch. Pro-mesolegs of males with a ventral adhesive patch in pro and mesotarsus. Receptacle of spermatheca subcylindrical shaped, without a noticeable border from pump; pump with a little appendage at apex. Internal sac of the median lobe with four sclerites.

Holotype male. ( Fig. 1 ). Color. General bright green: antennal calli yellowish; clypeus, labrum, distal onethird of mandibles, and maxillary palpi chestnut. Antennomeres chestnut, darker towards the apex, all surface of antennomere1 principally green. Disc and lateral margins of pronotum green, tinged with yellowish brown. Scutellum amber. Tarsi chestnut. Venter: metasternum and abdomen yellowish brown.

Head ( Figs. 10–11 ) Vertex finely and sparsely punctuate, deeply depressed above the antennal calli; antennal calli rounded, distinctly convex, as wide as the antennal sockets; supracallinal sulcus and midfrontal sulcus deeply impressed; supra-antennal sulcus barely distinct; antennal sockets closed to the anterior margin of eyes, separated by more than one-fourth the interocular space. Frontal ridge moderately raised in lateral view, ten long, thin setae situated below and lateral to the external antennal sockets; anterofrontal ridge not separated from frontal ridge. Genal space small, one-fourth the maximum ocular length. Antennae inserted at or scarcely below midline of eyes, extending a little more than half length of elytra; antennomere 2 short, antennomere 3 little longer than 2, antennomeres 2+3 together more than one-half the length of antennomere 4; antennomeres 3–10 elongate, similar in length, antennomere 11 apically acuminate. Clypeus with eight preapical setae. Labrum, ( Fig. 2 ) rectangular, anterior margin with a small central notch, with a row of six long setae, ventrally 11 thick setae in apical margin. Mandibles ( Fig. 12 ) five-toothed, teeth 3–5 visible on external face; tooth 3 fairly acute, 2.0 times longer than 4; tooth 4 acute, 2.0 times longer than 5; tooth 5, short, blunt at apex; inner margin of the mandible with 2 or 3 very short denticles; teeth 1-2 visible on internal face, acute; tooth 1 subequal to 5; tooth 2 longer than 4; mola wider than long, rows of fine grinding ridges well developed only in the external part, prosthecal fringe formed by long setae. Maxillae ( Figs. 3 , 13 ), with cardo apically broadened, with seven long setae; basistipes with three long setae situated in the latero-external margin; galea and lacinia with a fringe-like pilosity apically, galea surpassing the lacinia. Maxillary palpi with palpomere 1 subcuadrangular; palpomere 2 and 3 subcylindrical, the latter longer than palpomere 2; palpomere 4 subconical, strongly tapering apically; digitiform sensillum patch ( Fig. 14 ), on the externo-basal corner, formed by seven embedded sensilla. Labium ( Fig. 15 ) with four long setae between bases of palps; ligula bearing five setae. Labial palpi with palpomere 1 subrectangular; palpomere 2 subcylindrical, more than 2.0 longer than 3; palpomere 3 subconical with narrow base, subequal to 2.

Thorax. Pronotum slightly convex, subrectangular, 1.28 times wider than long, bifoveate; surface covered by minute punctures; widest at middle, anterior margin almost straight, lateral sides slightly expanded anteriorly, posterior margin arched; anterior callosity well produced, posterior callosity poorly developed, dentiform; each one bearing a long seta. Prosternum convex; intercoxal prosternal process thin, extended to about one-half of the procoxae. Scutellum triangular, smooth, rounded at apex. Metanotum transverse, wider than long; apodeme “d” ( Fig. 4 ) intersecting apodeme “c” at midpoint of “c”. Metendosternite ( Fig. 5 ) with stalk longer than wide; lateral arms, apically rounded, mesofurcal-metafurcal tendons poorly developed, inserted near middle of lateral arms. Hind wings ( Fig. 6 ) with veins RA, MP, CuA well sclerotized, whereas veins SC, CuA2 and AA scarcely sclerotized. Vein SC connected to RA at about three-fourths its length, radial cell, elongate, subtriangular; RP-MP2 almost reaching r4; RP-MP less than one-half the distance of MP 1-2, AA unbranched and connected to CuA3+4 more than one-half the distance from the origin of CuA; CuA2 attached to CuA, cubital anal cell closed, elongate; apical field with veins RP barely noticeable. Elytra with surface densely, irregularly punctuate, punctures somewhat coarser than on pronotum; elytra slightly wider than pronotum, HW/PW: 1.35, humeral calli rounded; greatest width near apical one third of elytra, EW/HW: 1.31; epipleura subvertical, basally broad, gradually narrowed apically; basal inner surface of elytra with an oval binding patch (Figs. 19–21) covered with stump-shaped spicules, apical end covered with small sharktoothshaped spicules; a fine submarginal ribbon (Fig. 22) along the surface near anterior margin covered with long, thin microspicules. All legs similar; with tibiae dorsally carinate, meso-metatibiae with short spurs. Tarsomere 1 of prolegs subrectangular, shorter than tarsomeres 2+3 together; tarsomere 1 of metalegs elongate, longer than tarsomeres 2+3 together; tarsal claws bifid, inner claw more than one-half the length of the outer. Promesolegs with a ventral adhesive patch (Figs. 16–18) covering almost all the length of protarsus, and approximately one-half on mesotarsus.

FIGURES 16–24. Diabrotica wartensis Cabrera & Sosa-Gómez (16) Male proleg, detail of tarsomeres, ventral view. (17) Male 1st. tarsomere, detail of adhesive patch. (18) Male mesoleg, 2nd tarsomere, detail of adhesive patch. (19) Elytron, detail of binding patch. (20) Binding patch, stump-shaped spicules. (21) Detail of binding patch, surface covered with stump-shaped spicules and sharktooth-shaped spicules on distal area. (22) Detail of submarginal ribbon along the surface near anterior margin, covered with thin microspicules. (23) Median lobe, lateral view. (24) Median lobe, dorsal view. Abbreviations: ap, adhesive patch; bf, basal foramen; bp, binding patch; op, orificial plate; os, ostium; shs, sharktooth spicules; sr, submarginal ribbon; sts, stump spicules.

Genitalia. Median lobe ( Figs. 7 , 23) evenly curved in lateral view, apex bent ventrally with a small projection; orificial plate (Fig. 24) elongate, apically acute; ostium wide, with a pair of well developed triangular lobes attached to the side. Internal sac ( Fig. 8 ) with four sclerotized plates.

Measurements (n= 12): Body length: 4.71–5.43 mm (0 = 5.15 ± 0.31), eL: 0.49–0,60 mm (0 = 0.53 ± 0.03), OD: 0.40–0.62 (0 = 0.54 ± 0.16), GL: 0.09–0.13 (0 = 0.11 ± 0.03), AD: 0.10–0.26 (0 = 0.17 ± 0.06), A1: 0.36–0.40 mm (0 = 0.37 ± 0.02), A2: 0.10–0.16 mm (0 = 0.13 ± 0.02), A3: 0.13–0.2 mm (0 = 0.16 ± 0.02), A4: 0.42–0.46 mm (0 = 0.44 ± 0.02), PL: 0.92–1.15 mm (0 = 1.06 ± 0.07), PW: 1.35–1.50 mm (0 = 1.44 ± 0.06), EL: 3.56–4.12 mm (0 = 3.88 ± 0.26), EW: 2.31–2.7 mm (0 = 2.57 ± 0.04), AD/OD: 0.25–0.39 (0 = 0.31 ± 0.06), GL/eL: 0,16–0,25 (0 = 0.20 ± 0.06), PW/PL: 1.28–1.46(0 = 1.35 ± 0.08), HW/PW: 1.33–1.35 (0 = 1.33 ± 0.01), EW/HW: 1.22–1.40(0 = 1.31 ± 0.04).

Female. The specimens examined are similar in color and sculpturing to the males. Body slightly larger. Antennomeres 2 and 3 subequal in length, together from more than one-half to three-fourths the length of antennomere 4. Interocular distance smaller in relation to males. Genal space one-fourth or more the maximum length of the eye. Pronotum wider than in males. All legs without a ventral adhesive patch covering surface of tarsomere 1.

Genitalia ( Fig. 9 ). Sternite 8 weakly sclerotized, apodeme (tignum) slender, slightly curved posteriorly. Styles (vaginal palpi) subcylindrical, rounded at apex with 10 setae. Vagina + bursa copulatrix large, undivided, with sclerotized area in the posterior part; spermathecal duct uncoiled, with a short sclerotized portion inserted basally in the receptacle; spermathecal gland elongate. Receptacle of spermatheca subcylindrical shaped, not well distinguishable from pump; pump curved, with a little appendage at apex.

Measurements (n = 13): Body length: 4.82–5.89 mm (0 = 5.24 ± 0.42), eL: 0.46–0.49 mm (0 = 0,53 ± 0.02), OD: 0.59–0.69 mm (0 = 0.65 ± 0.03), GL: 0.09–0.13mm (0= 0.12 ± 0.02), A1: 0.29–0.46 mm (0 = 0.36 ± 0.05), AD: 0.13 –0,19 mm (0 = 0,15 ± 0,02), A2: 0.13–0.16 mm (0 = 0.14 ± 0.02), A3: 0.13–0.19 mm (0 = 0.16 ± 0.02), A4: 0.33–0.39 mm (0 = 0.37 ± 0.01), PL: 0.92–1.15 mm (0 = 1.04 ± 0.10), PW: 1.35–1.68 mm (0 = 1.57 ± 0.16), EL: 3.46–4.46 mm (0 = 4.13 ± 0.07), EW: 2.57-3 mm (0 = 2.74 ± 0.19), AD/OD: 0.18–0.30 (0 = 0.23 ± 0.04), GL/eL: 0,20–0,34 (0 = 0.28 ± 0.01), PW/PL: 1.37–1.68 (0 = 1.49 ± 0.11), HW/PW: 1.23–1.39 (0 = 1.30 ± 0.06). EW/HW: 1.22–1.37(0 = 1.31 ± 0.07).

Intraspecific variation. This is a relatively uniform species, where the main differences are associated with color. In some species green fades to yellowish brown, but bright green is the prominent color of well preserved or live specimens. The degree of green of the elytra, thorax, head (except vertex) varies from lime green to bright green, but in some cases the head varies toward yellowish brown. The vertex and antennal calli are frequently the lightest areas of the head, and vary from bright green to yellowish. Labrum and mouthparts vary from chestnut to dark brown. Antennae (except the basal antennomere) unicolorous, vary from cinnamon to dark brown. The external face of femora vary to yellow, and tarsomeres to bright green.

Etymology. The name of this species refers to its site of discovery, Warta, Londrina Co. in Parana State.

Biological Notes. Adults of D. wartensis have been found in the north Paraná state region, on Cucurbita maxima Duchense and after the description of the species on Helianthus annuus L.

Geographic range This species is only known from the locality of Warta (S 23º 11´068” W 51º 10’ 206”, Datum WGS84), Londrina, Paraná State, Brazil.

Type material. Holotype: male, BRAZIL: Paraná: Warta, Londrina-PR, 14-I-2004, on Cucurbita maxima Duchense (Cucurbitaceae) , A. Micheli leg. Paratypes: 2 males and 4 females, with the same locality, date and collector of the holotype; 3 males and 4 females, same locality and collector except for listed dates: 2 females, 07-VI-2004, 2 males and 2 females, 12-0 I-04, and 1 male, 21-I-04 ( MZSP).

Molecular differentiation. COI sequences obtained in this study were deposited in the GenBank database (National Center for Biotechnology information, Bethesda, Maryland) under accession numbers EF 183512 and EF 183513 . DNA, and ITS-1 sequences under accession numbers EF 183514 and EF 183515 . Sequencing of the partial cytochrome oxidase subunit I ( COI) mitochondrial amplicon revealed an average size of 707 bp. The average base frequencies were A = 28.9%, C = 17.6%, G = 16.9%, and T = 36.5% for the entire amplification product. The mtDNA sequence corresponds to positions between 1,490 and 2,199 of the Drosophila melanogaster sequence encoding the cytochrome oxidase I gene, partial cds (GenBank accession U37541 , NC_001709). Of the 452 characters, 39 (8.6%) were variable, 286 were constant and 127 (28%) were parsimony informative.

Pairwise comparisons of the nucleotide sequence of the COI between D. wartensis and other Diabroticites resulted in divergence that ranged from 14.5% for D. nummularis to 20.1% for D. porracea ( Table 1 ).

For the ITS-1 PCR amplified product revealed an amplicon of 659 bp on average. The average base frequencies for this region were A=31.8%, C=13.9%, G=20.5%, and T=33.7%. The sequencing of the rDNA of D. wartensis resulted in an amplified product of an average size of 659 bp. The DNA sequences correspond to positions between 40 and 748 of the Diabrotica balteata sequence encoding the 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, complete sequence; and 5.8S ribosomal RNA gene, partial sequence (GenBank accession AF195200 ). Among all species considered, of 797 total characters, 156 characters were parsimony informative and 518 were constant. Divergences between D. wartensis and other Diabrotica species ranged from 13% to 16% when it was compared to D. balteata and to D. virgifera complex subspecies, respectively (Table 2).

Parsimony analysis of the ITS-1 region including all the sequences available, at the moment, in the Gen- Bank produced one most parsimonious tree (CI = 0.934; RI = 0.940, Fig. 25 ). Separate analysis of sequences of the ITS-1 region yielded a different overall topology. D. wartensis clustered with D. undecimpunctata howardi and D. balteata from fucata clade, in a well supported group, with 90% bootstrap value. However, phylogenetic analysis of the COI gene analyzed separately provide a strict consensus tree (CI=0.464; RI=0.453, Fig. 26 ) with similar topology to that obtained with the entire data set and different than that obtained with ITS-1 data.

The entire data set for the combined analysis contained 1,269 nucleotides, 472 from the COI mitochondrial DNA and 797 from the nuclear ITS-1 region. The total number of parsimony informative sites in the entire combined data set was 208 (16%). The analysis resulted in seven equally parsimonious trees (CI = 0.602; RI = 0.544).

In addition to the inferred relationships, a pragmatic approach consist on the potential to develop diagnostic tools based on the restriction pattern from the fragment length polymorphism ( RFLP) of the sequences obtained here. RFLP patterns in agarose or polyacrilamide gels of polymerase chain reaction products might be used to distinguish species and strains ( Roehrdanz et al. 2003).

Figures

FIGURE 1. Diabrotica wartensis Cabrera & Sosa-Gómez, male, dorsal habitus.

FIGURE 25. Cladogram of the strict consensus tree based on the ITS- 1 nuclear region generated by parsimony analysis as implemented Phylogenetic Analysis Using Parsimony (PAUP 4.0) and heuristic search algorithm. Bootstrap analysis of 1000 replicates was performed on the data using Parsimony method. Numbers shown on the branches are bootstrap percentages derived from parsimony. Only values of 50 % or greater are shown.

FIGURE 26. Cladogram of the strict consensus tree based on COI mitochondrial data (452 characters) generated by parsimony analysis as implemented Phylogenetic Analysis Using Parsimony (PAUP 4.0). Bootstrap analysis of 1000 replicates was performed on the data using Parsimony method using the heuristic search algorithm. Numbers shown on the branches are bootstrap percentages derived from parsimony. Only values of 50 % or greater are shown.

FIGURES 10 – 15. Diabrotica wartensis Cabrera & Sosa-Gómez (10) Head, frontal view. (11) Head, lateral view. (12) Mandible, external face. (13) Maxilla, dorsal view. (14) Maxilla, detail of digitiform sensillum. (15) Labium, ventral view. Abbreviations: ac, antennal callus; afr, anterofrontal ridge; bs, basistipes; dgs, digitiform sensillum; ds, ditistipes; fr, frontal ridge; ga, galea; lc, lacinia; lg, ligula; mo, mola; mp, maxillary palp; prm, prementon; sm, setose membrane; th 3, tooth 3; th 4, tooth 4; th 5, tooth 5.

FIGURES 2 – 9. Diabrotica wartensis Cabrera & Sosa-Gómez (2) Labrum, dorsal view. (3) Maxilla, ventral view. (4) Metanotum. (5) Metendosternite, dorsal view. (6) Hind wing. (7). Median lobe, lateral view. (8) Median lobe, detail of apex and internal sac, dorsal view. (9) Female genitalia. Abbreviations: a, metanotal ridge a; AA, anal anterior vein; b 1, metanotal ridge b 1; b 2; c, metanotal ridge c; CuA, cubitoanal vein; CuA 3 + 4, cubitoanal vein 3 + 4; d, metanotal ridge d; mg, median groove; MP 1 - 2, medial posterior vein 1 - 2; os, ostium; pu, pump; RA, radial vein; RAc, radial cell; re, receptacle; RP-MP 2, radial posterior-medial posterior vein 2; SC, subcostal vein; sc 1, sclerite 1; sc 2, sclerite 2; sc 3, sclerite 3; sc 4, sclerite 4; ssd, sclerotized spermathecal duct; tg, tignum; tl, triangular lobe; va-bu, vagina-bursa copulatrix; vg, vaginal palpi. Scale bars = 0.1 mm.