Description of the Larva of Notaticus fasciatus - Kelly Miller Lab

SYSTEMATICS

Description of the Larva of Notaticus fasciatus (Coleoptera: Dytiscidae) Associated with Adults Using DNA Sequence Data KELLY B. MILLER,1 YVES ALARIE,2

AND

MICHAEL F. WHITING3

Ann. Entomol. Soc. Am. 100(6): 787Ð797 (2007)

ABSTRACT Third instars of the diving beetle Notaticus fasciatus Zimmermann (Coleoptera: Dytiscidae) are described for the Þrst time, representing the Þrst description of larvae from Aubehydrini, the only remaining tribe of Dytiscinae unknown as larvae. The description is based on larvae suspected to be N. fasciatus collected in French Guiana. The specimens were associated with adults by analyzing an 806-bp fragment of cytochrome c oxidase I. Adults of N. fasciatus and 19 other species were included representing all tribes and most species of Dytiscinae found in northern South America with multiple representatives of some for a total of 33 terminals. Data were analyzed using parsimony and p-distances. The parsimony analysis resulted in a single tree. The unknown larva grouped with adult specimens of N. fasciatus (bootstrap 100%). Distance between them averaged 0.01 (0.01Ð 0.02) compared with 0.02 (0 Ð 0.06) between other conspeciÞcs and 0.13 (0.05Ð 0.17) between different species. N. fasciatus larvae differ from other Dytiscinae in the following characters: 1) head capsule subtriangular; 2) occipital suture absent; 3) anterior margin of frontoclypeus convex; 4) dorsal margin of foramen magnum straight; 5) dorsal pair of stemmata unmodiÞed; 6) antennomere I, maxillary palpomere I, and labial palpomeres I and II not subdivided; 7) maxillary palpomere II subdivided; 8) ligula absent; 9) galea well developed; 10) natatory setae present on dorsal margin of coxae, absent along ventral margin of trochanters and tarsi; 11) tarsal claws not serrate; 12) abdominal tergites fully developed; and 13) urogomphus well developed, shorter than last abdominal segment, and without natatory setae. KEY WORDS Neotropical, larva, diving beetle

The Neotropical genus Notaticus Zimmermann (Coleoptera: Dytiscidae), the only genus in the tribe Aubehydrini, is a rarely collected and phylogenetically unique taxon. It occurs in shallow lentic habitats with dense vegetation throughout lowland South America from Colombia to northern Brazil to northern Argentina. The genus was originally described in the Hydaticini (Dytiscinae) by Zimmermann (1928). It also was later described as Aubehydrus Guignot (1942) and placed in its own subfamily, Aubehydrinae Guignot. This placement was based mainly on the lack of an externally visible scutellum, as in most Hydroporinae and Laccophilinae, combined with character states that exclude it from those two subfamilies. Guignot (1949) described the male and illustrated the male genitalia (both original descriptions did not include males) and also provided additional distribution information. Spangler (1973a) synonymized Aubehydrus with Notaticus, described the male again in detail, and summarized the knowledge of the taxon. Tre´ m1 Corresponding author: Department of Biology, Museum of Southwestern Biology, University of New Mexico, Albuquerque, New Mexico 87131 (e-mail: [email protected]). 2 Department of Biology, Laurentian University, Ramsey Lake Road, Sudbury, Ontario P3E 2C6, Canada. 3 Department of Integrative Biology, Brigham Young University, Provo, UT 84602.

ouilles and Bachmann (1981) added new distribution records, and Wolfe (1985) included the genus as an outgroup in his analysis of Hydroporinae. Miller (2000) placed the taxon back within the subfamily Dytiscinae based on a cladistic analysis of adult characters, and, Þnding it to have a phylogenetically unique position, placed it in its own tribe, Aubehydrini. This was further conÞrmed by Miller (2001, 2003) based on morphological and molecular data. Although Ribera et al. (2002) also found Notaticus to be derived within Dytiscinae, they found evidence for it nesting within Hydaticini where it was originally placed by Zimmermann (1928). Finally, Garcia and Navarro (2001) recently described a putative new species from Venezuela, N. confusus Garcia & Navarro. Placement of Notaticus within Dytiscinae is well founded (Miller 2000, 2001; Ribera et al. 2002; Miller 2003). However, its relationships within the subfamily are less convincingly determined. It has been placed within Hydaticini (Zimmermann 1928, Ribera et al. 2002) as sister to (Hydaticini ⫹ (Eretini ⫹ Aciliini)) (Miller 2000, 2001) and as sister to Aciliini (Miller 2003). One of the problems in placing Notaticus is the lack of a larval description for the taxon. Larvae of all other tribes of Dytiscinae (and most of the genera) have been described, and larval characters contribute considerable information for classiÞcation within the

0013-8746/07/0787Ð0797$04.00/0 䉷 2007 Entomological Society of America

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ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA

subfamily. Knowing the character states present in Notaticus larvae likely would contribute to a better understanding of the groupÕs phylogenetic relationships. Two unusual larval specimens of a dytiscine were collected by K.B.M. in wetlands at the margins of the Kaw River in northern French Guiana. These specimens are clearly larvae of Dytiscinae based on 1) presence of subdivided antennomeres and maxillary palpi; 2) presence of a maxillary galea; 3) the femur, tibia and tarsus with a row of natatory setae along the anteroventral margin; 4) the urogomphus one-segmented; and 5) the lateral margins of abdominal segments VII and VIII with a fringe of natatory setae. However, the larvae lacked characteristic features that would place them in one of the tribes of Dytiscinae known as larvae. Thus, these larvae, which have a unique character combination not known for any dytiscine tribe and are in the right size range (Notaticus specimens are among the smallest Dytiscinae), were suspected to be the unknown Aubehydrini. The specimens were collected during a short collecting trip to a remote area, and their identity was not suspected until they were examined in the laboratory. Thus, rearing them was impossible, and although the evidence was compelling that they belong to Notaticus, further conÞrmation was desirable. An earlier attempt to associate larvae and adult specimens of Dytiscidae was very successful (Miller et al. 2005). Therefore, we used DNA sequence data to provide additional evidence for identifying these larvae to species. In forthcoming projects, we intend to address more thoroughly the larval structure of the Dytiscinae and its contribution to knowledge of dytiscine phylogeny. Therefore, we comment only brießy on the phylogenetic implications of N. fasciatus larval structure. Materials and Methods Materials. Two putative instar IIIs of N. fasciatus were collected from a single locality in French Guiana (Table 1). Two adult specimens collected in Bolivia were available for comparison (Table 1). Also included were representatives of the other New World dytiscine taxa: Megadytes Sharp, Cybister Curtis, Dytiscus Linnaeus, Eretes Laporte, Thermonectus Dejean, Hydaticus Leach, Graphoderus Dejean, and Acilius Leach (Table 1). The cladogram was rooted using a cybistrine (the Neotropical Cybister puncticollis Brulle´ ), because Cybistrini is sister to the remaining Dytiscinae (Miller 2003). Two or more specimens of several species were sequenced to examine intraspeciÞc variation in the DNA. DNA Sequences. Methods for DNA analysis closely followed those of Miller et al. (2005). DNA was extracted using the DNEasy kit (QIAGEN, Valencia, CA) protocol for animal tissue. DNA from adults was extracted by making a slit along the side of the thorax, and thoracic muscle tissue was removed and placed in extraction buffer with the remaining portions of these

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specimens retained as vouchers. Vouchers are deposited in the frozen tissue collections of K.B.M. and Brigham Young University (Provo, UT). DNA from larvae suspected to be Notaticus was extracted by making a slit along the side of the thorax, placing the entire specimen in buffer, and retrieving after incubation for vouchering. Larvae extracted in this way are cleared and suitable for slide mounting. Larval vouchers are deposited in the collection of Y.A. An 806-bp portion of the mitochondrial gene cytochrome c oxidase I (COI) was ampliÞed and sequenced. This gene fragment has been used for phylogenetic and population analyses of diving beetles (Ribera et al. 2001; Ribera et al. 2003a, 2003b, 2004; Balke et al. 2004) and previous associations of larvae and adult diving beetles (Miller et al. 2005) The primer pair C1-J-2183 (“Jerry”) and TL2-N-3014 (“Pat”) (Simon et al. 1994) was used to amplify this region. The fragment was ampliÞed using polymerase chain reaction (PCR) on a DNA Engine DYAD Peltier thermal cycler (Bio-Rad, Hercules, CA). AmpliÞcation conditions were 95⬚C (12 min) for one cycle, 94⬚C (1 min), 48 Ð52⬚C (1 min), 64 Ð 68⬚C (1 min) for 40 cycles, and 64 Ð 68⬚C (7 min) for one cycle. Contamination was mediated using negative controls. Fragments produced from PCR were examined using gel electrophoresis. Fragments were puriÞed using Montage PCR96 Cleanup kit (Millipore, Billerica, MA) and cycle sequenced using ABI Prism BigDye version 3 (Applied Biosystems, Foster City, CA) by using the same primers used to amplify. Sequencing reaction products were puriÞed using Sephadex G-50 medium and sequenced using an ABI 3730xl DNA analyzer (DNA Sequencing Center, Brigham Young University). The gene region was sequenced in both directions. Resulting sequence data were examined and edited using the program Sequencher (Genecodes 1999), and sequences were submitted to GenBank (see Table 1 for GenBank numbers). DNA Sequence Analysis. Analysis for this project was similar to that done by Miller et al. (2005). Alignment of sequences was based on conservation of codon reading frame in Sequencher (Genecodes 1999). Sequences were not length variable in these taxa, and the resulting alignment was unambiguous. Data were analyzed using parsimony and the program NONA (Goloboff 1995) as implemented using the “heuristics” menu option in the program WinClada (Nixon 2002) and the following settings: hold 5000 trees total (“h 5000”), 40 replications (“mu*40”), 40 trees held per replication (“h/40”), and multiple TBR ⫹ TBR (“max*”) swapping. Trees were examined and characters mapped under different optimizations by using WinClada. Bootstrap values were calculated using WinClada and the options “1000 replicates,” “10 search reps,” “1 starting tree per rep,” and “DonÕt do max*.” Bremer values (Bremer 1994) were calculated using NONA and the commands “hold 10000,” “suboptimal 10” and “bsupport 10”. Pairwise distances were calculated with the program MEGA 2.1 (Kumar et al. 2001) by using the “Calculate distances” option and “Nucleotide: p-distance” model option for distances.

November 2007 Table 1.

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Species and specimens used in analysis, collection locality information, and GenBank accession numbers for COI sequences Species

Acilius semisulcatus Aub 关1兴 A. semisulcatus 关2兴 A. semisulcatus 关3兴 A. semisulcatus 关4兴 Cybister puncticollis (Brull) 关1兴 C. puncticollis 关2兴 Dytiscus dauricus Gebler 关1兴 D. dauricus 关2兴 Eretes sticticus (Linnaeus) Graphoderus liberus (Say) Hydaticus aruspex (Clark) 关1兴 H. aruspex 关2兴 H. rimosus Aub H. subfasciatus Laporte 关1兴 H. subfasciatus 关2兴 H. xanthomelas (Brull) 关1兴 H. xanthomelas 关2兴 Hyderodes shuckardi (Hope) 关1兴 H. shuckardi 关2兴 Megadytes fraternus Sharp 关1兴 M. fraternus 关2兴 Notaticus fasciatus Zimmermann 关1兴 N. fasciatus 关2兴 N. fasciatus 关larva兴 Thermonectus basillaris (Harris) T. circumscriptus (Latreille) T. leprieurii Balfour-Browne T. margineguttatus (Aub) T. marmoratus (Hope) T. nobilis Zimmermann T. succinctus (Aub) 关1兴 T. succinctus 关2兴 T. variegatus (Laporte)

Collection locality (K.B.M. voucher no.)

GenBank no.

USA: New York, Tompkins Co., pond 10 km S Caroline, 23 Oct. 2000, KB Miller, leg. (Acse7) Alberta: University Pond, 54⬚ 43⬘ N 113⬚ 16⬘ W 25 Sept. 2001, E Goth-Birkigt, leg. (Acse153) Canada: Alberta, W5mer. Twp 33 Rge 7 SE ⬇8 km SE Bearberry, 8 Sept. 2002, J Bergsten, leg. (Acse161) Canada: Alberta, W 4mer. Twp66 Rge22 s. 27 NE N Athabasca, 1 Sept. 2002, J Bergsten, leg. (Acse162) Bolivia: Dpto. Beni, Prov Cercado 9.5 km N Trinidad, 14⬚ 46⬘ 34⬙ S 64⬚ 58⬘ 00⬙ W 17 June 1999, KB Miller, leg. (Cypu21) Same as above (Cypu110) Canada: Alberta, W4mer. Twp77 Rge14, 4 Sept. 2002 J Bergsten, leg. (Dyda175) Japan: Hokkaido, Karimun, Moraseppu Town, 24 Aug. 2000, J Bergsten, leg. (Dyda177) Namibia: Tsaobis NP, temp pools, 22 Feb. 2002 F Johansson, leg. (Erst189) USA: New York, Tompkins Co., Ringwood Preserve 19 Sept. 2000, KB Miller, leg. (Grli69) USA: New York, Schuyler Co., Texas Hollow SWA 6 Sept. 2000, KB Miller, leg. (Hyar68) Sweden: Ang. Orsten, 25 Aug. 2002, J Bergsten, leg. (Hyar187) Costa Rica: Guanacaste Prv., Barra Honda N. Park, Quebrada La Palma, 12 Jan. 2004, Short & Lebbin, legs. (Hyrm335) Bolivia: Dpto. Sta Cruz, Prov. Chiquitos 2.7 km S San Jose, 17⬚ 52⬘ 20⬙ S 60⬚ 44⬘ 28⬙ W 333 m 27 June 1999, KB Miller, leg. (Hysu23) French Guiana: Kaw Mountain Res., forest pools nr. Amazone Lodge, 8Ð18 Feb. 2005, 04⬚ 32⬘ 57.8⬙ N 52⬚ 12⬘ 49.7⬙ W, KB Miller, leg. (Hysu391) Bolivia: Dpto. Sta Cruz, Prov Velaso, cattail marsh, 1.5 km SE San Ignacio, 24 June 1999, KB Miller, leg. (Hyxa67) Same as above (Hyxa114) Australia: Victoria, roadside pool ⬇20 km W Cowwarr 38⬚ 00⬘ 52⬙ S 146⬚ 32⬘ 03⬙ E, 07 Nov. 2000, KB Miller, leg. (Hdsh88) Same as above (Hdsh104) USA: Florida, Collier Co., JaneÕs Scenic Rd & Stewert Fakahatchee Strand Preserve State Park, 22 Mar. 2000 (Mefr55) Paraguay, Dpt. Alto Paraguay, Ea. Choroveca, 29 Nov. 2002 (Mefr300) Bolivia: Dpto. Beni, Prov. Cercado 9.5km N Trinidad 14⬚ 46⬘ 34⬙ S 64⬚ 58⬘ 00⬙ W 17 June 1999, KB Miller, leg. (Nofa52) Same as above (Nofa112) French Guiana: Kaw Mountain Res., Kaw River boat launch 04⬚ 29⬘ 54.8⬙ N 052⬚ 03⬘ 05.6⬙ W, KB Miller, leg. (Nofa392) USA: New York, Schuyler Co., Texas Hollow SWA 12 Sept. 2002, 42⬚ 24.73⬘ N 076⬚ 47.58⬘ W, 358 m, J Bergsten, leg. (Thba210) Peru: Madre de Dios, boat landing ⬇20km S InÞerno nr Puerto Maldonado, 14 Dec 2003, KB Miller, leg. (Thci257) Peru: Rio Tambopata, Explorers Inn, jnct Rio Tower 12⬚ 50.208⬘ S 069⬚ 17.603⬘ W, 10 Dec. 2003, KB Miller, leg. (Thle259) Same as above (Thmg260) USA: Arizona, Pima Co. stock tank, ⬇6 km S Arivaca 29 Apr. 2000 31⬚ 31⬘ 15⬙ N 011⬚ 16⬘ 44⬙ W, 1,088 m, KB Miller, leg. (Thma13) Peru: Madre de Dios, boat landing ⬇20 km S InÞerno nr Puerto Maldonado, 14 Dec. 2003, KB Miller, leg. (Thno256) Bolivia: Dpto. Sta Cruz, Prov Chiquitos, 2.2 km E San Jose artiÞcial cattle pond, 26 June 1999, KB Miller, leg. (Thsu265) Bolivia: Dpto. Sta Cruz, 20 km W San Jose, 17⬚ 44.903⬘ S 061⬚ 04.202⬘ W, 13 Jan. 2004, Svenson et al., legs. (Thsu267) Peru: Rio Tambopata, Explorers Inn, jnct Rio Tower 12⬚ 50.208⬘ S 069⬚ 17.603⬘ W, 10 Dec. 2003, KB Miller, leg. (Thva258)

DQ275307 DQ431202 DQ431203 DQ431204 DQ431201 DQ431200 DQ431205 DQ431206 DQ431207 DQ431208 DQ431209 DQ431210 DQ431211 DQ431212 DQ431213 DQ431214 DQ431215 DQ431216 DQ431217 DQ431218 DQ431219 DQ431220 DQ431221 DQ431222 DQ431223 DQ431224 DQ431225 DQ431226 DQ431227 DQ431228 DQ431229 DQ431230 DQ431231

Multiple specimens from the same species are followed by a number in brackets.

The p-distance is the uncorrected proportion of sites where two sequences differ. It is calculated by dividing the number of nucleotide differences by the total number of nucleotides (Kumar et al. 2001). Larval Analysis. Instar III specimens were disarticulated and mounted on standard glass slides with HoyerÕs medium. All measurements were made with a

compound microscope equipped with a micrometer eyepiece. The part to be measured was adjusted so that it was, as nearly as possible, parallel to the plane of the objectives. The characters and terms used in the morphometric analysis are mainly those used in previous articles dealing with larval morphology of the Dytiscidae (e.g.,

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Fig. 1. Single most parsimonious cladogram derived from parsimony analysis of COI sequences from Dytiscidae specimens (length ⫽ 1,125, CI ⫽ 37, RI ⫽ 62). Branch lengths are proportional to number of nucleotide changes mapped using “fast” optimization in WinClada. Numbers below branches are bootstrap values (percentage). Branches without numbers have bootstrap values ⬍50%. Numbers below branches are Bremer supports.

Alarie et al. 2000, 2001, 2002) and some are deÞned as follows. Head Length (HL). Total head length, including the frontoclypeus measured medially along the epicranial stem. Head Width (HW). Maximum width measured at level of the stemmata. Length of Frontoclypeus (FCL). Distance from the apex of the nasal to the posterior margin of the ecdysial suture. Occipital Foramen Width (OcW). Maximum width measured along the dorsal margin of the occipital foramen. Length of Antenna. Derived by adding the length of each individual antennomere; comparison among antennomeres was made using the capital letter A with a number corresponding to the segment considered (e.g., A1 for antennomere I); because A2 and A3 are each secondarily subdivided into two subsegments (A2.1 and A2.2; A3.1 and A3.2) (Fig. 5), their relative lengths were derived by adding the length of each individual subsegment. A3⬘ is used as an abbreviation for the lateral elongation of antennomere III. Length of Maxillary Palpus. Derived by adding the length of each individual palpomere (e.g., MX1 for palpomere I). Because MX1 and MX2 are each secondarily subdivided into two subsegments (MX1.1 and

MX1.2; MX2.1 and MX2.2) (Fig. 6), their relative lengths were derived by adding the length of each individual subsegment. Length of Maxillary Galea. Maximum length measured from apex of the galea to the margin of the maxillary stipes. Length of Palpifer. Maximum length measured along lateral margin. Length of Labial Palpus. Derived by adding the length of each individual palpomere (e.g., LB1 for palpomere I). Length of Legs. Derived by adding the length of each individual segment including the longest claw; the length of each segment was taken at the longest point except for the trochanter, which includes only the proximal portion (the length of distal portion is included in the femoral length). Dorsal Length of Last Abdominal Segment (LLAS). Includes the whole sclerite measured dorsally along the midline from the anterior margin to the posterior margin. The individual measurements deÞned above were used in calculating several ratios intended to characterize the body shape. Most of the ratios used in this article are similar to those used in previous reports dealing with larval morphology of the Dytiscidae (cf., supra), and as such, they are not deÞned herein.

0.13 0.13 0.12 0.12 0.11 0.12 0.12 0.13 0.13 0.13

0.13 0.13 0.12 0.12 0.11 0.12 0.12 0.13 0.14 0.13

Acilius semisulcatus 关1兴

0.01 0.01 0.14 0.14 0.13 0.13 0.14 0.12 0.13 0.13 0.13 0.11 0.11 0.13 0.13 0.14 0.14 0.16 0.15 0.15 0.15 0.15

Acilius semisulcatus 关2兴

0.01 0.01 0 0.15 0.15 0.13 0.14 0.14 0.12 0.13 0.13 0.13 0.11 0.11 0.13 0.13 0.14 0.14 0.17 0.15 0.16 0.15 0.15

0.13 0.13 0.12 0.13 0.11 0.12 0.12 0.13 0.13 0.13

0.01 0.14 0.14 0.13 0.13 0.14 0.12 0.13 0.13 0.12 0.10 0.10 0.13 0.13 0.14 0.14 0.16 0.15 0.15 0.15 0.15

A. semisulcatus 关3兴

A. semisulcatus 关4兴

0.13 0.13 0.12 0.12 0.11 0.12 0.12 0.13 0.13 0.13

0.14 0.14 0.13 0.13 0.14 0.12 0.13 0.13 0.13 0.10 0.11 0.13 0.13 0.14 0.14 0.17 0.15 0.15 0.15 0.15

Values ⱕ 0.05 indicated with bold.

A. semisulcatus 关2兴 A. semisulcatus 关3兴 A. semisulcatus 关4兴 C. puncticollis 关1兴 C. puncticollis 关2兴 D. dauricus 关1兴 D. dauricus 关2兴 E. sticticus G. liberus H. aruspex 关1兴 H. aruspex 关2兴 H. rimosus H. subfasciatus 关1兴 H. subfasciatus 关2兴 H. xanthomelas 关1兴 H. xanthomelas 关2兴 H. shuckardi 关1兴 H. shuckardi 关2兴 M. fraternus 关1兴 M. fraternus 关2兴 N. fasciatus 关1兴 N. fasciatus 关2兴 N. fasciatus (larva) S. bakewelli T. basillaris T. circumscriptus T. leprieurii T. margineguttatus T. marmoratus T. nobilis T. succinctus 关1兴 T. succinctus 关2兴 T. variegatus

Cybister puncticollis 关1兴

0.14 0.14 0.14 0.13 0.13 0.16 0.12 0.15 0.15 0.14

0 0.13 0.12 0.16 0.13 0.15 0.16 0.15 0.13 0.13 0.13 0.13 0.15 0.15 0.14 0.13 0.17 0.17 0.17

C. puncticollis 关2兴

0.14 0.14 0.14 0.13 0.13 0.16 0.12 0.15 0.15 0.14

0.13 0.12 0.16 0.13 0.15 0.16 0.15 0.13 0.13 0.13 0.13 0.15 0.15 0.14 0.13 0.17 0.17 0.17

Dytiscus dauricus 关1兴

0.13 0.12 0.12 0.12 0.12 0.12 0.10 0.13 0.13 0.13

0.03 0.14 0.12 0.12 0.13 0.12 0.12 0.12 0.12 0.12 0.13 0.13 0.16 0.15 0.15 0.14 0.14

D. dauricus 关2兴 0.12 0.12 0.11 0.12 0.11 0.12 0.11 0.12 0.12 0.13

0.14 0.12 0.13 0.13 0.12 0.12 0.12 0.12 0.12 0.14 0.14 0.15 0.15 0.15 0.14 0.15

Eretes sticticus 0.13 0.12 0.10 0.13 0.12 0.12 0.11 0.12 0.12 0.13

0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.16 0.16 0.16 0.15 0.15 0.14 0.14

Graphoderus liberus 0.12 0.13 0.11 0.12 0.12 0.14 0.11 0.12 0.12 0.13

0.14 0.14 0.11 0.11 0.11 0.12 0.13 0.14 0.14 0.14 0.13 0.14 0.14 0.14

Hydaticus aruspex 关1兴 0.13 0.12 0.11 0.12 0.12 0.12 0.11 0.13 0.13 0.12

0.06 0.11 0.11 0.10 0.12 0.12 0.14 0.14 0.16 0.14 0.15 0.14 0.14

H. aruspex 关2兴 0.13 0.13 0.12 0.13 0.12 0.13 0.11 0.13 0.14 0.12

0.12 0.11 0.11 0.12 0.12 0.14 0.14 0.15 0.15 0.15 0.15 0.15

H. rimosus 0.13 0.11 0.10 0.12 0.11 0.12 0.10 0.12 0.12 0.11

0.08 0.09 0.08 0.09 0.13 0.13 0.15 0.13 0.13 0.13 0.13

H. subfasciatus 关1兴 0.11 0.11 0.09 0.11 0.11 0.12 0.11 0.11 0.12 0.11

0.01 0.07 0.07 0.13 0.13 0.15 0.13 0.13 0.12 0.12

H. subfasciatus 关2兴 0.12 0.11 0.08 0.11 0.11 0.12 0.11 0.12 0.12 0.11

0.07 0.07 0.12 0.12 0.15 0.13 0.13 0.12 0.12

H. xanthomelas 关1兴 0.11 0.10 0.09 0.11 0.09 0.11 0.10 0.11 0.11 0.12

0.01 0.12 0.12 0.14 0.12 0.13 0.12 0.12

H. xanthomelas 关2兴 0.11 0.10 0.09 0.11 0.09 0.11 0.10 0.11 0.11 0.12

0.12 0.12 0.14 0.13 0.13 0.13 0.12 0.12 0.15 0.13 0.14 0.14 0.15 0.14 0.14 0.14 0.15

0 0.15 0.15 0.15 0.15 0.14

Hyderodes shuckardi 关1兴

Pairwise distances between taxa in analysis expressed as proportion of nucleotide differences (p-distances)

0.12 0.15 0.13 0.14 0.14 0.15 0.14 0.14 0.14 0.15

0.15 0.15 0.15 0.15 0.14

H. shuckardi 关2兴

Table 2.

N. fasciatus 关2兴

Notaticus fasciatus 关1兴

M. fraternus 关2兴

Megadytes fraternus 关1兴 0.15 0.15 0.15 0.15 0.15 0.16 0.14 0.15 0.15 0.15

0.14 0.13 0.13 0.14 0.13 0.14 0.12 0.13 0.13 0.14

0.13 0.13 0.12 0.14 0.13 0.14 0.13 0.13 0.14 0.13

0.13 0.13 0.11 0.14 0.13 0.13 0.13 0.13 0.13 0.12

0.05 0.17 0.16 0.17 0.16 0.01 0.17 0.16 0.02 0.01

N. fasciatusL 关larva兴 0.13 0.13 0.11 0.13 0.12 0.13 0.13 0.13 0.13 0.12

Sandracottus bakewelli 0.11 0.10 0.12 0.11 0.12 0.10 0.11 0.11 0.13

Thermonectus basillaris 0.08 0.08 0.07 0.09 0.06 0.08 0.08 0.10

T. circumscriptus 0.09 0.08 0.10 0.08 0.07 0.08 0.09

T. leprieurii 0.09 0.11 0.07 0.10 0.10 0.11

T. margineguttatus 0.09 0.05 0.07 0.07 0.10

T. succinctus 关2兴

T. succinctus 关1兴 T. nobilis

T. marmoratus

0.09 0.10 0.07 0.10 0.07 0 0.10 0.09 0.11 0.11

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Chaetotaxic Analysis. Primary and secondary setae and pores were distinguished on the head appendages and legs where possible. Because no system of nomenclature for primary sensilla of the Dytiscinae has been proposed, the primary setae and pores were coded according to the system proposed for the genus Lancetes Sharp, because it has been suggested to share a sister-group relationship with members of the subfamily Dytiscinae (Alarie et al. 2002). Setae are coded using two capital letters corresponding to the Þrst two letters of the name of the structure on which the seta is located (AN, antenna; CO, coxa; FE, femur; MX, maxilla; LA, labium; TA, tarsus; TI, tibia; TR, trochanter) and a number. Pores are coded in a similar manner except that the number is replaced by a lower case letter. First instars of the genus Lancetes are characterized by the presence of additional setae on the legs. Because no Þrst instar was available for this study, we were unable to differentiate additional setae (i.e., those setae evolved secondarily in the Þrst instar; generally restricted to a genus or a tribe) from the secondary setae (i.e., those added throughout the ontogenetic development of the larva). Accordingly, additional setae are included in the count of secondary setae. Color. Description of color is given from ethanolpreserved specimens. Color in Dytiscidae larvae sometimes changes because of ethanol preservation, but color pattern is not ordinarily altered. Results The cladistic analysis resulted in a single most parsimonious tree of length 1125 (CI ⫽ 37, RI ⫽ 62) (Fig. 1). The unknown larva is unambiguously resolved in a group with the adult specimen of N. fasciatus (bootstrap value ⫽ 100%, Bremer support ⬎10). The distance between nonconspeciÞcs in the analysis averaged 0.13 (range 0.05Ð 0.17, SD ⫽ 0.02) (Table 2). The average distance between members of the same species was 0.01 (range 0 Ð 0.06, SD ⫽ 0.02) (Table 2). The average distance among adults of N. fasciatus and the unknown larva was 0.01 (range 0.01Ð 0.02, SD ⬍0.01) (Table 2). Larvae of Dytiscinae Third instars of Dytiscinae can be distinguished from those of other Dytiscidae by the following combination of characters: 1) antennomeres II and III (Fig. 5), maxillary palpomere III (Fig. 6) secondarily subdivided; 2) femora, tibiae, and tarsi with a row of natatory setae along dorsal margin (Fig. 10); 3) femora and tibiae with a row of natatory setae along ventral margin (Fig. 11); and 4) abdominal segments VII and VIII with a row of natatory setae along lateral margins (Figs. 2 and 12).

Figs. 2–4. Notaticus fasciatus Zimmermann, instar III. (2) Habitus, dorsal view (color pattern not represented). Scale bar, 0.50 mm. (3 and 4) Head capsule: (3) dorsal aspect; (4) ventral aspect (color pattern not represented). Scale bar ⫽ 1.00 mm.

following combination of characters: 1) head capsule subtriangular (Figs. 2Ð 4); 2) occipital suture absent (Figs. 2 and 3); 3) anterior margin of frontoclypeus convex (Figs. 2 and 3); 4) dorsal margin of foramen magnum nearly straight (Figs. 2 and 3); 5) dorsal pair of stemmata of normal size (Figs. 2 and 3); 6) antennomere I (Fig. 5), maxillary palpomere I (Fig. 6), and labial palpomeres I and II (Figs. 7 and 8) not secondarily subdivided; 7) maxillary palpomere II secondarily subdivided (Fig. 6); 8) ligula absent (Figs. 7 and 8); 9) galea well developed (Fig. 6); 10) natatory setae present on dorsal margin of coxae (Fig. 10), absent along ventral margin of trochanters (Figs. 10 and 11); 11) tarsal claws not serrate (Figs. 10 and 11); 12) abdominal tergites fully developed (Fig. 2); and 13) urogomphus well developed, shorter than LLAS, without natatory setae (Figs. 2 and 12).

Larvae of Aubehydrini (Figs. 2–12)

Description of Instar III of N. fasciatus Zimmermann (Figs. 2–12) (n ⴝ 2)

Third instars of Aubehydrini can be distinguished from those of other tribes of the Dytiscinae by the

Color. Head capsule. Dorsal surface as in Fig. 3. Head appendages. Predominantly black except A2 and

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Figs. 5–6. Notaticus fasciatus Zimmermann, instar III, head appendages: (5) Antenna, ventral aspect, A1, A2, A3 ⫽ antennomeres I, II, and III; A3⬘ ⫽ lateral elongation of antennomere III. (6) Maxilla, ventral aspect, MX1, MX2, MX3 ⫽ maxillary palpomeres I, II, and III. Numbers and lowercase letters refer to primary setae and pores, respectively. Scale bars ⫽ 0.50 mm.

apex of A4, yellowish. Thorax. Terga dark yellow to piceous, with a variable number of black longitudinal stripes more apparent along lateral margins. Legs. Predominantly yellow except for a narrow piceous band at about mid-length of each article. Abdomen. Terga similar to thoracic terga; segment VIII slightly darker. Urogomphus. Predominantly piceous to black, yellow in middle for a short distance. Head (Figs. 2–9). HL ⫽ 2.24 mm; HW ⫽ 1.35 mm; FCL ⫽ 0.84 mm. Head capsule (Figs. 3 and 4). ßattened, subtriangular, longer than broad (HL/HW ⫽ 1.66), slightly constricted at level of occipital region, maximum width at about level of stemmata, HW/ OcW ⫽ 3.89; ecdysial suture well developed, coronal suture ⬇0.63⫻ HL; occipital suture absent; frontoclypeus slightly convex mesally, 0.38⫻ HL, not extending mesally beyond level of lateral lobes [⫽adnasalia]; apical margin of frontoclypeus with numerous spatulate setae (lamellae clypeales of Bertrand 1972); gular suture not visible, parietale meeting ventrally; ocularium present, stemmata visible ventrally and subdivided into two vertical series; tentorial pits visible ventrally on each side of middle at about

mid-length; occipital foramen deeply emarginate ventrally. Antenna (Fig. 5). Four-segmented, shorter than HW (length of antenna/HW ⫽ 0.69); A1 ⬎ A2 ⫽ A3 ⬎ A4, A2/A3 ⫽ 1.06, A3⬘/A4 ⫽ 1.00; antennomere III with a ventroapical spinula. A2 and A3 secondarily subdivided into two articles, A2.1 ⬍ A2.2, A3.1 ⬍ A3.2. Mandible (Fig. 9). Falciform, inner dorsal margin with row of short setae apically, inner ventral margin minutely dentate at about mid-length, short and robust, 2.93⫻ width, ⬇0.40⫻ length of HL; mandibular channel present. Maxilla (Fig. 6). Stipes subrectangular, elongate, subequal to length of palpus; cardo and galea present, lacinia absent; galea 0.85⫻ length of palpomere I; palpifer similar in shape to palpomeres, 0.42⫻ length of palpomere I; palpus three-segmented, shorter than antenna (length of antenna/length of maxillary palpus ⫽ 1.56); MX1 ⫽ MX2 ⫽ MX3; length of MX3/length of MX2 ⫽ 1.19; MX2 and MX3 secondarily subdivided into two articles, MX2.1 ⬍ MX2.2, MX3.1 ⬍ MX3.2. Labium (Figs. 7 and 8). Prementum subtrapezoidal, width slightly greater than length, ligula absent; palpus two-segmented, shorter than maxillary palpus (length of maxillary palpus/length of

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Figs. 7–9. Notaticus fasciatus Zimmermann, instar III, head appendages: (7 and 8) Labium: (7) dorsal aspect; (8) ventral aspect. (9) Mandible, ventral aspect. Numbers and lowercase letters refer to primary setae and pores, respectively. Scale bars ⫽ 0.50 mm.

labial palpus ⫽ 1.55), LB2 0.90⫻ length of palpomere I. Chaetotaxy and porotaxy. Head capsule with several secondary setae; parietale with ⬇20 short lateral spine-like setae; head appendages without secondary setae except A1 with four secondary setae along outer lateral margin (Fig. 5) and mandible with several minute secondary setae (Fig. 9). Thorax. Pronotum subrectangular to subtrapezoidal dorsally, about same length as meso- and metanotum combined; metanotum subequal to mesonotum in length, both slightly wider than pronotum; terga with an anterotransverse carina and with sagittal line well

deÞned; thoracic venter membranous; mesopleural region with spiracular opening on each side. Legs (Figs. 10 and 11). Five-segmented; length of metathoracic legs greatest, ⬇1.10⫻ length of prothoracic legs, and 2.56⫻ HW; meta[coxa ⫽ femur ⫽ tibia ⬎ trochanter ⫽ tarsus]; tarsus with two claws, posterior claw slightly shorter than anterior claw; anterior metathoracic claw 0.26⫻ length of metatarsus; spinulae strongly developed on ventral margin of proand metatarsus, absent on mesotarsus. Chaetotaxy and porotaxy. Position and number of secondary setae as in Table 3; natatory setae present along dorsal margin of

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Figs. 10–11. Notaticus fasciatus Zimmermann, instar III: Metathoracic leg; (10) anterior surfaces; (11) posterior surfaces. CO, coxa; FE, femur; TR, trochanter; TI, tibia; TA, tarsus. Numbers and lowercase letters refer to primary setae and pores, respectively. Scale bar ⫽ 0.50 mm.

coxa, femur, tibia and tarsi and along ventral margin femur and tibia. Abdomen (Figs. 2 and 12). LLAS ⫽ 1.21Ð1.39 mm. Eight-segmented, dorsally sclerotized; segments IÐVI membranous ventrally, segments VIIÐVIII completely sclerotized; terga IÐVI with an anterotransverse carina and a sagittal line; maximum body width at level of segments III and IV; segments VII and VIII subrectangular to subcylindrical; LLAS/HW ⫽ 0.65; segments IÐVII each with pair of spiracles; siphon reduced in size, slightly sinuate along lateral margins. Chaetotaxy and porotaxy. Secondary tergal setae present; segments VII and VIII each with a row of natatory setae along lateral margin. Urogomphus. Total length of urogomphus ⫽ 0.87 mm; one-segmented, without secondary subdivisions, 0.67⫻ length of LLAS, 0.67⫻ length of HW. Chaetotaxy and porotaxy. Secondary setae absent. Discussion The evidence presented here strongly suggests that the unknown larvae belong to Notaticus. The se-

quenced specimen is resolved in a clade with adults of N. fasciatus in the parsimony analysis (bootstrap ⫽ 100, Bremer ⬎10), and there are low distances between it and the adult N. fasciatus (0.01Ð 0.02). Divergence patterns between the included dytiscines are similar to values for other insects (e.g., Hebert 2004) and mitochondrial gene divergences between animals in general (Avise 2000). There are two described species in the genus from northern South America, N. fasciatus and N. confusus Garcia & Navarro. The differences between these two species in adult characters are exceptionally minor, consisting of N. confusus being larger, having more spines on the posterior tibia, and having the prosternal process more strongly longitudinally impressed (Garcia and Navarro 2001). The male genitalia of the two species (Þgured by Guignot 1949, Spangler 1973b, Garcia and Navarro 2001) seem to be identical. We suspect that the two names refer to the same species. Nevertheless, it is possible that the larvae hypothesized here to be N. fasciatus actually belong to another species of Notaticus (such as N. confusus). The larva is resolved as the sister to a clade made up of the two adults, rather than nesting

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Table 3. Number of secondarya setae on the legs third instar of Notaticus fasciatus Zimmermann

Segment ProCO ProTR ProFE

ProTI

ProTA

MesoCO MesoTR MesoFE

Fig. 12. Notaticus fasciatus Zimmermann, larva instar III, abdominal segments VII (distal part) and VIII, dorsal aspect. Scale bar ⫽ 0.50 mm.

among them. This weakens the conclusion that the larvae belong to N. fasciatus in light of the possibility of other species of Notaticus occurring in northern South America. However, the two adults sequenced here were collected in Bolivia, and the distance in COI between them and the larval specimen (0.02) is well within what would be expected for specimens of conspeciÞc dytiscids (Table 2; Miller et al. 2005). Therefore, we suspect that the results are based on population differences rather than interspeciÞc variation. Nevertheless, future researchers encountering possible different or new species of Notaticus can test our hypothesis by sequencing and analyzing additional specimens. Larvae of N. fasciatus are characterized by the absence of an occipital suture (Fig. 3) and smaller size (although several species of Thermonectus from South America are similar in size to N. fasciatus). Placement of Notaticus within the Dytiscinae is evident based on larval morphology. Third instars of N. fasciatus share with other members of the Dytiscinae: 1) secondary subdivision of the antennomeres II and III as well as of the maxillary palpomere III (Figs. 5 and 6); 2) indentation of the ventral margin of the foramen magnum (Fig. 4); 3) the presence of a row of natatory setae along the dorsal margin of the femora, tibiae, and tarsi as well as along the ventral margin of femora and tibiae (Figs. 10 and 11); and 4) the presence of a dense row of natatory setae along the

Sensillar series

Secondary setae (n ⫽ 1)

AD(NS) V Total Pr Di Total AD AV(Sp) AV(NS) PD(NS) PV Total AD AV(Sp) AV(NS) PD(NS) PV Total AD AV PD(NS) Total AD(NS) V Total Pr Di Total AD AV(Sp) AV(NS) PD(NS) PV Total

9 0 9 2 1 3 11 7 16 9 10 53 9 10 19 18 4 60 5 0 12 17 8 1 9 2 1 3 10 11 15 11 9 56

Segment MesoTI

MesoTA

MetaCO MetaTR MetaFE

MetaTI

MetaTA

Sensillar series

Secondary setae (n ⫽ 1)

AD AV(Sp) AV(NS) PD(NS) PV Total AD AV PD(NS) Total AD(NS) V Total Pr Di Total AD AV(Sp) AV(NS) PD(NS) PV Total AD AV(Sp) AV(NS) PD(NS) PV Total AD AV PD(NS) Total

10 13 20 21 5 69 5 10 16 33 11 5 16 2 1 3 9 13 13 9 8 52 9 16 21 18 11 75 5 5 15 25

AD, anterodorsal; AV, anteroventral; CO, coxa; D, dorsal; Di, distal; FE, femur; NS, natatory setae; PD, posterodorsal; Pr, proximal; PV, posteroventral; Sp, spine-like; TA, tarsus; TI, tibia; TR, trochanter; V, ventral; n, number of specimens studied. a Including additional setae (cf. Material and Methods).

lateral margin of both abdominal segments VII and VIII (Figs. 2 and 12). Within the Dytiscinae, the absence of secondary subdivision of the antennomere I (Fig. 5), of the maxillary palpomere I (Fig. 6), and of both the labial palpomeres I and II (Figs. 7 and 8) suggests a relationship of Notaticus with members of Hydaticini, Eretini, and Aciliini. The absence of a ligula (Figs. 7 and 8), the secondary subdivision of maxillary palpomere II (Fig. 6), the lack of indentation along the dorsal margin of the foramen magnum (Fig. 3), the normal size of the dorsal pair of stemmata (Fig. 3), the presence of a row of natatory setae along the dorsal margin of the coxae (Fig. 10), and the lack of natatory setae along the ventral margin of the tarsi (Figs. 10 and 11) suggest a close relationship of Notaticus with members of the Hydaticini. Acknowledgments KBM especially thanks J. Cryan, J. Urban and T. McCabe, collecting partners in French Guiana and elsewhere. Support for KBM and MFW came in part from National Science Foundation grants #DEB-0073088, #DEB-9983195, #DEB0329115 and #DEB-0515924. Financial support for YA was

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provided by the Natural Sciences and Engineering Research Council of Canada in the form of an operating research grant.

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