Larvae and a new species of Ancyronyx Erichson, 1847 (Insecta, Coleoptera, Elmidae) from Palawan, Philippines, using DNA sequences for the assignment of the developmental stages

Abstract Ancyronyx montanus sp. n. is described based on adults and larvae, matched using their cox1 DNA sequence data. Larvae of six additional species of Ancyronyx Erichson, 1847 were also described here for the first time, aided by cox1 or cob data: Ancyronyx helgeschneideri Freitag & Jäch, 2007, Ancyronyx minerva Freitag & Jäch, 2007, Ancyronyx patrolus Freitag & Jäch, 2007, Ancyronyx procerus Jäch, 1994, Ancyronyx punkti Freitag & Jäch, 2007, Ancyronyx pseudopatrolus Freitag & Jäch, 2007. Ancyronyx procerus is newly recorded from the Philippines by a larval specimen from Busuanga island. The new species and larval stages are described in detail and illustrated by digital and SEM images. A key to the Ancyronyx larvae of Palawan and an updated checklist of Philippine Ancyronyx is provided.


Introduction
The genus Ancyronyx, usually referred to as spider water beetle, belongs to the predominantly aquatic riffle beetle family Elmidae Curtis, 1830 (Coleoptera), subfamily Elminae Curtis, 1830. Ancyronychini Ganglbauer, 1904 has been erected exclusively for this genus which is known from North America and Southeast Asia. The adults have extremely long legs and strong claws as an adaptation to their riverine habitats. Elmidae are often highly sensitive to water pollution and are therefore of great value as bioindicators (e.g. Moog & Jäch 2003;Hilsenhoff 1982). This requires, however, taxonomic knowledge and appropriate identification tools. Ancyronyx larvae were unknown until Brown (1972) illustrated those of the North-American species A. variegatus (Germar, 1824). Philippis (1997) published the first data on the life cycle and growth of this species. He successfully used head capsule width to assign the respective instar stage to the larvae.
Publications dealing with molecular data of Elmidae are very rare and DNA data are only available for one Ancyronyx species, A. procerus (Čiampor & Ribera 2006. The first Ancyronyx species from the Philippines was recorded and described by . Subsequently, seven new species were added , Freitag & Jäch 2007) and more new species await description. Currently, the Philippine province of Palawan is very well sampled with regard to Elmidae due to the AQUA Palawana Program (http://aquapalawana.nhm-wien.ac.at) conducted by the first author for more than 10 years. The copious collection of Ancyronyx specimens, both adult and larvae, retrieved by this taxonomic research initiative gave us the opportunity to study the larvae of this genus taxonomically for the first time.

Taxon sampling
The larvae examined were partly collected by standardized methods for ecological studies such as colonization and drift sampling during a survey in 2000 / 2001 (Freitag 2005). Such samples are referred to by the letters "C" (colonization sample) or "D" (drift sample), while manual samples are indicated by "M" at the end of a collection label. This variety of methods appeared to be more successful to retrieve rare species (Freitag, 2008). Most larval material of Ancyronyx helgeschneideri was obtained this way. However, the collected samples were preserved in formalin, thus the materials are consequently not suitable for molecular-genetic analysis.
All materials from more recent surveys have been retrieved by means of manual collection from submerged wood debris or through the use of a fine-meshed hand net. This material was preserved in absolute ethyl alcohol and thus, it was suitable for genetic sequencing. The best manual sampling was possible in permanent small to medium sized rivers in forested area (Fig. 1).
The label codes for the sampling sites of the first author are arbitrary. They do not follow any temporal or spatial pattern, except for the fact, that eventually varying small letters following a common code number refer to different sections of the same water system.

Phylogenetic analysis
Podelmis viridiaenea Jäch, 1982 (Elmidae: Elminae: Elmini) from Sri Lanka was used as an outgroup. Two additional sequences of Ancyronyx procerus were retrieved from GenBank: DQ266500.1, DQ266511.1 (Čiampor and Ribera 2006). Sequences were edited and aligned in CLUSTALW (Thompson et al. 1994) using BIOEDIT version 7.0.5.2. (Hall 1999 and default parameters. Phylogenetic analyses were conducted with MRBAYES vers. 3.1.2 (Ronquist and Huelsenbeck 2003) using the GTR (General Time Reversible) model (Tavaré 1986) with default priors starting with random trees with three heated and one cold Markov chains. The analysis was run by 1,000,000 generations, 7,501 trees were sampled after the first 25% of samples from the cold chain have been discarded as burnin. Branch support for the Bayesian trees was assessed with posterior probabilities determined via the 50% majority rule consensus. This "quick" analysis was sufficient for the task of detecting which larva might be associated with which adult.

Morphological analysis
Scanning electron microscope (SEM) images were obtained using a ZEISS EVO 50 XVP at SMTD. The specimens were coated with gold using two samples each if enough material was available. Those taxa of which only a single or few specimens have been available were only vacuum dried, but not gold-coated prior to scanning. This resulted in lower quality of the micrographs, but has kept the specimen´s surface natural.
Digital habitus photographs were taken with a NIKON SMZ800 stereo microscope with digital photo adapter NIKON DS-Fi1 (unit in DLSU). These photographs were taken at various focus layers and were subsequently combined using COMBINEZM software (Hadley 2008) to retrieve images with sufficient depth of focus. The same system was used for the dissection of adult specimens and the detailed material examination.
Examination, biometric measuring and imaging of dissected parts were conducted using a NIKON Eclipse 600 microscope with a ZEISS AxioCam MRc5 digital camera (unit in SMTD). The morphological details of larvae are described from external view as they are usually visible without dissection, if not stated opposite. The listing of examined material includes the head width (in mm) of all measured larvae.
Morphological terminology used herein mainly follows the Elmidae chapter of the recently published Handbook of Zoology / Coleoptera (Kodada and Jäch 2005

DNA sequence analysis
Alignment of the cox1 data and trimming ambiguous bases at the 3' and 5' ends yielded a matrix of 770 bp. None of the sequences contained indels. The sequence of the larvae of A. pseudopatrolus had ambiguous six positions in-between, that were coded as 'N's. The sequence divergence between Ancyronyx species was between 3.3.% (25 positions different: A. patrolus / A. montanus ) and 18% (139 positions different: A. helgeschneideri / A. montanus). The highest divergence with another taxon (17%-20%) was with the outgroup species in all cases.
All adults and larvae could be matched unambiguously. Sequences of adult and larva of the same species from the same locality or island showed low difference of positions in most species (2-3 positions different, 0.2-0.4% divergence). Ancyronyx pseudopatrolus would be within this range when assumed that all unidentified positions are not varying between adult and larva, which is likely as these positions appear to be conserved sites in Ancyronyx. Sequence samples of the same species, but from different islands showed slightly higher divergence of 0.6%-2.1% (5-16 positions). A 50% majority rule consensus trees based on cox1 data is illustrated in Fig. 2.
The sequencing of cox1 of the one and only available alcohol-preserved larva of Ancyronyx helgeschneideri failed and is consequently not included in the phylogenetic analyses. However, we were able to amplify the cob sequence of that larval specimen and match it with its adult stage. Their aligned cob sequences of 378 bp were identical except for two positions of synonymous exchange of a base (0.5% divergence). In Ancyronyx procerus six positions of the cob sequences varied between adult and larval specimens by synonymous changes (1.6% divergence). Both species varied in 61 positions of their cob sequence (16% divergence).
Variation between larval instars. Almost the entire material studied belongs to the final instar stage. The two presumably prefinal instar specimens do not vary conspicuously from the description above. The legs appear slightly broader and shorter in relation to the body and their setae patterns are slightly different.
HW 0.32 mm; entire larva about 3.0 mm long. Body elongate, broader than that of A. minerva, but very similar in the external characters, except for the following: Posterolateral projections (Fig. 12A) of abdominal segments IV-VIII distinctly overreaching posterior segment margins (approximately as long as the squamose setae at posterior segment margins).
Variation between larval instars. The few available prefinal instar specimens vary from the description above by relatively slenderer thoracic and abdominal segments, the relatively longer posterolateral projections, the distinctly shorter and broader legs and fewer setae on tibiae and femora.
Larval differential diagnosis. The species can most easily be distinguished from A. minerva which looks superficially most similar by the colour pattern of ventral head, pronotum and last abdominal segment and the longer median crest of the first abdominal segment venter.
Distribution. Known from Palawan island. Remarks. This species was originally described based on a single male exemplar. As additional material including females have been collected since then we will provide a short diagnosis of the female characters and sexual dimorphisms. Furthermore, the aedeagus (Fig. 13A) is figured in here as SEM micrograph, for the detailed diagnosis see Freitag & Jäch (2007), pp. 41 & 46;Figs 12a, b. Adult female diagnosis. Ovipositor as in Fig. 13B. Total length c. 500 µm. Stylus comparably stout as in A. punkti (see Freitag & Jäch, 2007, Fig. 13d), slightly conical towards base (broader apically), slightly outwards directed. Coxite long and slender as in A. patrolus (comp. Freitag & Jäch, 2007, Fig. 11d); setae rather short, peg-like, apically rounded, not acute, most densely dispersed at coxite apex; mesal coxite margin moderately pubescent; basal portion c. half as long as distal portion, with slightly more acute and pointed setae than those at distal portion, most densely set at proximal and lateral margins. Valvifer about as long as coxite; fibula enlarged and curved inwards at proximal end.

Ancyronyx pseudopatrolus
Secondary sexual characters. Sternite VIII in female (Fig. 13C) overall very similar to that of A. punkti, with median strut apically widened and truncate; posterior portion with disc densely covered with small, inconspicuous setae; posterior margin with moderately long trichoid setae. Sternite VIII in male weakly sclerotized, median strut distinctly shorter than in female. Ventrite 5 in female (Fig. 13D) overall very similar to that of A. punkti, subtriangular, with small and only slightly elevated lateral projec- tions. Ventrite 5 in male (see Freitag & Jäch, 2007, Fig. 12d) suboval, stouter and with large and distinctly elevated lateral projections. Tergite VIII in female (Fig. 13E) longer than broad; condyles more or less straight; asperities only conspicuous laterally.
Larval diagnosis (based on 6 th instar). Colour (Fig. 5) somewhat similar to that of A. minerva and A. punkti, but differs slightly in the dorsal and leg colour patterns. Anterior yellow pronotal band very broad, reaching up to anterior 0.4, sublaterally extended posteriad (area of explanate lateral pronotal gutter). Meso-and metanotum and abdominal segments with paler entire posterior margin or entirely dark brown (except for paler posterolateral projections). Colour patterns of head and abdominal segment IX as in A. punkti. Legs yellowish pale except for dark distal tibia area around claw insertion.
HW c. 0.29 mm; entire larva about 3.0 mm long. Body shape as in A. punkti, except for the following characters: All abdominal posterolateral projections (Fig. 14A) very prominent, distinctly overreaching posterior segment margins. Setiferous tubercles at dorsal site very prominent and protruding (Figs 14A, B). Ventral side densely covered with asperities and scattered setiferous tubercles (Fig. 14E).
Sublateroposterior portions and anteriomedian sclerites of thoracic venters with setiferous tubercles (Fig. 14F); remaining ventral areas densely covered with asperities. Legs (Fig. 14G) as those of A. punkti. Abdomen (Figs 5; 14A, H-I) with dorsosagittal carinae at the posterior portions of segments I-VIII and most distinct at the almost entire segment IX (Fig. 14I). Squamose setae at posterior rim of segments I-VIII broken off in specimen figured under SEM, but generally developed (Fig. 5). Sagittal ridge of ventral sclerite of segment I longer than 1/2 of segment length. Apex of segment IX slightly emarginate. Operculum (Fig. 14H) medially deeply impressed, rugulose, with few setae at disk.
Variation between larval instars. For this species, quite a number of prefinal instar specimens is available. They vary from the final instar description by the overall paler colour, relatively longer posterolateral projections, relatively shorter and broader legs and fewer setae on tibiae and femora.
Larval differential diagnosis. The species can most easily be distinguished from its congeners A. minerva and A. punkti by the colour pattern of the anterior pronotum and the dorsal posterior margins of thoracic and abdominal segments and the more distinctly developed, protruding tubercles and of the dorsal crest of the abdominal segment IX.
Distribution. Only known from few rivers in central Palawan.  Fig. 6: almost entirely darkbrown; antennae, area of explanate lateral thoracic and abdominal gutter, lateral head portions and legs (except for darkened area around claw insertation) pale brown or yellowish. Mouthparts and surrounding portions of head capsule almost black. Without pale pattern at dorsal thoracic and abdominal segments; only apex of abdominal segment sightly paler. Ventral side pale brown.

Ancyronyx patrolus
HW 0.32 mm; entire larva up to 3.5 mm long. Body shape as in A. minerva, except for the following: dorsosagittal carinae at the posterior portions of abdominal segments IV-VIII and the entire length of segment IX very distinct (Figs 15A, B). Setiferous tubercles at dorsal side very prominent and protruding (Figs 15A, B) as in A. pseudopatrolus. Ventral side smoother, with scattered setae (Fig. 15I).
Head as in Fig. 6 and Figs 15C-F, similar to that of A. pseudopatrolus. Glabrous area with stemmata slightly exposed. With few short, acuminate setae on each side and a dorsolateral pair of long double setae (Fig. 15C). Frontal suture U-shaped ( Pro-, meso-and metathorax as in Figs 6,15G and Fig. 15H. Dorsal thoracic segments with conspicuous small round signa (arranged as in Fig. 15G) and a distinct sagittal line (sagittal line partly fused with signa). Thoracic venters (Fig. 15H) rather smooth as in A. minerva. Legs (Figs 15H-J) similar to those of A. minerva; inner side of tibiae and femora with longitudinal rim of spinous setae (Fig. 15J); all other parts with scattered squamose setae. Abdomen (Figs 6; 15A, B, I, K) with distinct dorsal sagittal line and slightly elevated surrounding portions in segments I-IV. Dorsosagittal carinae at the posterior portions of abdominal segments IV-VIII distinctly elevated, somewhat drop-shaped and densely covered with setiferous tubercles (Fig. 15B). Sagittal ridge of ventral sclerite of first segment distinctly shorter than 1/2 of segment length (Fig. 15I). Apex of segment IX emarginate. Operculum (Fig. 15K) elongately subtrapezoidal, medially moderately impressed, rugulose.
Variation between larval instars. Specimens of 4 th and 5 th instar vary only little from the final larval instar. The species typical dorsosagittal carinae of the posterior abdominal segments are very distinct. However, the emargination of the abdominal segment IX` apex is not conspicuous, the overall colour is paler, the thoracic and abdominal segments are relatively narrower, the legs are slightly shorter and broader and the setae on tibiae and femora are fewer and not well arranged in rims.
Larval differential diagnosis. The species can clearly be distinguished from other Palawan species by the lack of pale dorsal colour patches and its highly elevated dorsosagittal carinae which is most conspicuous at the posterior portion of abdominal segment VIII where it appears as a drop-shaped protuberance.
Distribution. Only known from Palawan and Busuanga. Colouration (Fig. 7) predominantly dark brown to black; legs pale brown to dark brown, (articulations slightly darker); claws and antennae pale brown (distal antennal segment darker); elytra without pale patches; ventral side slightly paler than dorsal, but still dark brown.
Legs slightly longer than body; pro-and mesocoxae large, globular; metacoxae only slightly protruding laterally; femora, tibiae, and tarsi (except distal tarsal segment) covered with elongate setiferous tubercles; tibiae distally with a distinct rim of setae; claws (Fig. 16G) well developed, rather gently curved; base of each claw with three teeth, distal one largest, basal one shortest.
Ovipositor (Fig. 16O). Total length c. 620 µm. Stylus slender, slightly bent outwards (partly broken off in specimen examined). Coxite long and slender, distal portion distinctly elongate, with several comparably long, lanceolate setae, most densely set at apex; mesal margin densely pubescent; basal portion c. half as long as distal portion, with same type of setae in distinct patterns as in Fig. 16O. Valvifer about as long as coxite; fibula (mesal, longitudinal sclerotisation) genus typical as in Fig. 16O.
Secondary sexual characters. Sternite VIII in female (Fig. 16P) with median strut apically widened, almost truncate, posterior portion slightly pubescent; sternite VIII in male weakly sclerotized, median strut distinctly shorter than in female. Ventrite 5 in female subtriangular (Fig. 16I), in male (Fig. 16J) shorter and suboval. Tergite VIII in female (Fig. 16Q) longer than broad; condyles more or less straight and prominent; reticulations only conspicuous laterally. Tergite VIII in male (Fig. 16R) broader than long, reticulation more developed than in female covering apical half; basal half patterned with lines of asperities; condyles not distinctly curved, overreaching anterior margin.
Adult differential diagnosis. A. montanus superficially resembles dark specimens of A. patrolus (Freitag & Jäch 2004 : Fig. 4), from which it can be easily distinguished by the larger size, slenderer body (EL/EW), the entirely reticulate pronotal surface that lacks any glabrous areas, the medially narrowed prosternal process and its genital characters. Fig. 8: very similar to that of A. patrolus, but overall darker and somewhat shiny; legs entirely pale brown to yellowish. Mouthparts and surrounding portions not distinctly darker than dorsal and ventral head portions. Without pale pattern at dorsal thoracic and abdominal segments; only apex of abdominal segment slightly paler. Ventral side pale brown.

Larval description (based on 6 th instar). Colour as in
HW 0.37-0.41 mm; entire larva up to 3.3 mm long. Body shape as in A. patrolus, except for the following: entire body more vaulted in cross section; dorsal sagittal area flat, with rather indistinct sagittal line, without carina at all thoracic and abdominal segments. Setiferous tubercles at dorsal side rather flat, not as elevated as in the other species (Fig. 17A), those of ventral side very flat and inconspicous (Fig. 17B). Setae originated from tubercles (Figs 17A-C) lanceolate to trichoid and distinctly longer than in the previous species. Posterolateral abdominal projections comparably small, attenuate, distinctly larger at anterior abdominal segments (Figs 8, 17C). Trichoid teeth not reaching middle of subsequent abdominal segment when that retracted.
Head (Figs 8, basically as in the previous species, but with the following varying characters: broadest posterior 0.25, laterally slightly convex. Glabrous area with stemmata not exposed. With few long, trichoid setae on lateral sides and one dorsolateral pair of long double setae (Figs 17D-F). Entire head more or less densely covered with squamose to fascicular setae originated from slightly elevated tubercles and additionally with several moderately long trichoid setae (Figs 17E-F). Frontal suture V-shaped. Fasciculate setae at subbasal fringe of clypeus very long, overreaching labrum (Fig. 17E); labrum with large fasciculate setae (Fig. 17E). Gula, maxillae and labium (Fig. 17F) almost as in A. minerva, but with few squamose setae and shorter maxillary and labial palpi. Mandibles as in Fig. 17E. Antennae (Fig. 17E) almost as in A. patrolus, scapus with large plumose setae.
Variation between larval instars. The two specimens of prefinal instar stage vary most conspicuously from the above description by the overall paler brown colour, the more conspicuous dorsal setiferous tubercles that let the pronotal signa appear clearly as well as the slightly crested (in cross-section subtriangular) abdominal segment IX.
Larval differential diagnosis. The species is easily distinguishable from other Palawan species by the lack of any dorsosagittal carinae or elevations, the dark, shiny dorsal colour, its rather large size and the rather shallow dorsal tubercles bearing comparably long, lanceolate to trichoid setae.
Distribution. Only known from the type locality in central Palawan and one site in northern Palawan (Fig. 20).
Etymology. The species is named in reference to the remote mountainous river habitats where it was exclusively recorded from. Larval description (based on 6 th instar). Colour (Fig. 9) predominantly brown; head distinctly darker to almost black dorsally; lateral head, antennal scape, anterior pronotal collar and legs whitish or yellowish pale. Entire ventral side (except for parts of genae), antennal pedicel, anterior margin of pronotum, claws, lateral abdominal projections, small medioposterior areas or entire posterior margin of thoracic and abdominal segments and a median middle portion of abdominal segment IX pale brown to yellowish.
HW c. 0.62 mm, entirely c. 3.7 mm long. Body flattened dorsoventrally, moderately vaulted dorsally, almost flat ventrally, with sagittal line (longitudinal groove from prothorax at least up to 5 th abdominal segment). Dorsal side moderately densely covered with setiferous tubercles (Fig. 18A). Ventral side smoother, with scattered setae and few setiferous tubercles (Fig. 18B). Retractable portions of body segments and pronotal collar without setae and tubercles (Fig. 18A). Lateral margins of abdominal segments I-VIII produced laterad forming posterolateral-directed conical projections (Fig. 18C). Projections increasing in size caudad, those of segment VIII c. 3.5 times as long as such on segment I. Rather inconspicuous spiracles present laterally on mesothorax and abdominal segments I-VIII.
Head (Figs 18A, D-F) subquadrate, partly retractable, distinctly prognathous, with three anterior-dorsad directed, pointed projections, one each side between antenna and clypeus and one at median frons (Fig. 18A). Frons rather glabrous, only with small and scattered setiferous tubercles. Stemmata arranged as single lateral spot in a glabrous area, slightly exposed. One irregular rim of moderately long setae at ventrolateral head margin (not visible in dorsal view). Frontal suture broadly Vshaped. Frontoclypeal suture uneven, but somewhat straight. Clypeus distally microreticulate, with protuberant anterior seam; without subbasal fringe of setae or tubercles. Ventral side (Fig. 18D) with few scattered setae and an obvious longitudinal crest each side lateral of gula and maxillae. Genae rugose, with asperities and scattered tubercles. Gula with rather inconspicuous asperities. Maxilla (Figs 18D, E) moderately broad; cardo stout, undivided; lateral portion with one median acuminate seta; stipes subrectangular, glabrous, with few short and one long latero-subapical trichoid setae; maxillary palpus ( Fig. 18E) four-segmented, approx. as long as stipes broad, distal segment smallest, cylindrical with several apical sensilla of various shape; predistal segment with lateroapical trichoid seta; galea and lacinia subequal in length and shape, approx. as long as palpus, apically with sensilla. Labium (Figs 18D, E) with broad (about 1.7 times of stipes) mentum (postmentum), broadest at basal half, with median groove most depressed posteriorly, with one pair of moderately long trichoid setae sublaterally at anterior 0.25, one subbasal pair each of spinose and trichoid setae and one pair of short apical lateral teeth. Submentum (prementum) short, transverse, apically convex, with sagittal ridge and one laterobasal pair of setae; ligula inconspicuous with various setae and pegs; labial palpi short, with short and stout palpifer; apical segment similar to that of maxillary palpi, preapical segment with lateral tuft (Fig. 18E). Mandibles not examined. Labrum c. 3 times as wide as long, anterior margin distinctly convex, with a subapical fringe of ramose setae and scattered trichoid and truncate (sensory) setae, proximal portion glabrous. Antenna (Fig. 18F) three-segmented, c. 1/2 as long as head. Peduncle short, about as long as broad, without (visible) dorsolateral fringe of branched trichoid setae; scape cylindrical, longer than pedicel and c. twice as long as broad, with few apical trichoid setae; pedicel cylindrically elongate, comparably short, only slightly longer than scape; flagellum and sensorium (broken off in figured specimen) subequal in length, slender, cylindrically elongate, c. five times as long as broad; apex of flagellum with inconspicuous elongate sensillum.
Legs (Figs 18G, H) stout (compared to larvae of the previous species and adults), similar in shape and length, with scattered trichoid sensilla mainly at femora and tibiae. Coxae large, subtrapezoidal; trochanter shorter, elongately subtrapezoidal, rather slender; femora subtrapezoidal, short; tibiae subcylindrical, broadest basal, distinctly narrower than femur, longer than other segments. Claws elongate, moderately bent, with one subbasal presumably trichoid tooth (broken or invisible in specimen examined). Abdomen (Figs 9; 18B, I, J). Segments I-VIII similar in shape, broadly subrectangular in dorsal view; terga with depressed sagittal line at least from 1 st up to 5 th segment. Retractable anterior portion with squamose asperities; posterior terga margins with a rim of squamose setae. Remaining median portions of terga more or less equally covered with setiferous tubercles. Ventral sclerites of segments I-VIII subrectangular, extendingly fused with pleural sclerites from 1 st to 8 th segment; posterior venter margins with a rim of squamous setae. Segment IX (Figs 18I, J) elongate, subconical (broadest subbasally), subtriangular in cross-section; apex broadly rounded, not emarginate; dorsal and lateral portions densely covered with setiferous tubercles; ventral side with scattered short trichoid setae and some long filiform setae sublaterally (most broken off in specimen figured in Fig. 18J). Operculum (Fig. 18I) subtrapezoidal to subtriangular, less than double as long as broad, medially depressed, rugose. Basal half with small longitudinal ridges and scattered sensilla; apical half with squamose asperities and a lateral rim of trichoid setae; the internally inserted pair of hooks rather small. Gill chamber with long, ramose gill tufts overreaching the opercular margin.
Variation between larval instars. The two final instar specimens available do not allow to draw conclusions about variations between the instars.
Larval differential diagnosis. The species resembles the previous ones only in very general characters, such as the presence of posterolateral appendages and the distribu-abdominal, meso-and metathoracic margins including projections as well as median portion of segment IX pale yellowish; anterior pronotal edges with conspicuous pale pattern that is extending mediad to disc; remaining parts of dorsal thorax and abdomen brown with indistinct pale patterns; the latter most conspicuous as yellowish spot at posterosagittal margin of all segments. Ventral side entirely pale, except for pale brown gula, maxillae, labium and ventral parts of genae.
HW c. 0.50 mm, entire larve up to 4.5 mm long. Body shape somewhat similar to that of A. procerus in the external characters, except for the following characters: spiracles distinctly larger, very prominent (Figs 10,19A); entire lateral margin with distinct long, trichoid setae (Figs 10,19F); tubercles at dorsal side more prominent (Figs 19A,B), but dorsal setae very short.
Head (Figs 19B-E) broadest subbasally, slightly conical anteriad, without median pointed projection at frons; pair of sublateral anterior projections between antenna and clypeus rather shallow and inconspicuous (Fig. 19B). Frons moderately densely covered with moderately large and equally dispersed setiferous tubercles; genae rugose, with scattered tubercles; lateral glabrous area with stemmata irregularly shaped. Antenna (Figs 19B-D) short, c. 1/3 as long as head. Scape short, as long as broad, with subapical fringe of stout sensilla; pedicel cylindrical, less than two times as long as scape and c. three times as long as broad, with few inconspicuous apical sensilla; flagellum and sensorium as in A. procerus. Labrum (Fig. 19C) c. 2.5 times as wide as long; lateroapical edges rounded; dorsal surface with tubercles, ramose setae and short trichoid setae. Ventral head (Figs 19D,E) with well-developed longitudinal crests. Maxilla (Figs 19D,E) almost as in A. procerus. Labium (Figs 19D,E) with mentum (postmentum) broadest in apical half, one pair of moderately long trichoid setae inserted sublaterally at anterior 0.25; some additional inconspicuous setae present at lateral margin in apical half; pair of apicolateral teeth slender, inserted at a distinct subapical crenation; submentum (prementum) divided.
Prothorax slightly broader than long; tergum with irregularly shaped signa in posterior half. Venter of pro-, meso and metathorax (Fig. 19F) similar to that in A. procerus, but anterior sclerites more oblique.
Legs as in Figs 19F, G and very similar to those in A. procerus. Abdomen (Figs 10; 19A, G-L) with terga slightly depressed groove along sagittal line at least from 1 st up to 4 th segment; venter almost glabrous, with scattered trichoid setae of different length; segment IX as in Figs 19J-L with emarginate apex; operculum ( Fig. L) glabrous, with rather inconspicuous basal ridges; disc without conspicuous asperities, covered with few scattered setae.

Discussion
We successfully used mitochondrial DNA sequencing to associate different life stages of beetles with each other, substantiated by morphological description of larvae. Use of DNA sequences has helped to avoid potential pitfalls, as for A. procerus samples where an unknown larva of a species, which was formerly not recorded in the area, occurred syntopically on the very same piece of wood debris with adults only of another species. This could have led to a misinterpretation as to which larva belongs to which adult.
The morphological species groups suggested by Freitag & Jäch (2007) were supported here by morphological characters of the larvae. Two main types are recognized. The Ancyronyx patrolus species group is characterized by smaller size, more vaulted larval body shape (in cross section) and rather small, caudal-directed posterolateral abdominal projections. The larvae of the A. variegatus species group are larger, depressed (in cross section), and display large, lateral-directed posterolateral projections.
The female and the larva of Ancyronyx minutulus Freitag & Jäch, 2007 still remain unknown to science. No additional material has been collected since the single holotype male was described despite intense sampling on Palawan Island including its type locality.