Contribution to the knowledge of larval chaetotaxy of the genus Enochrus Thomson: description of five New World species (Coleoptera: Hydrophilidae)

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Contribution to the knowledge of larval chaetotaxy of the genus Enochrus
Thomson: description of five New World species (Coleoptera: Hydrophilidae) Table I.Measurements (in mm) and ratios for different larval instars of described Enochrus species.Abbreviations: see Material and Methods section.(Fikáček, 2006;Byttebier & Torres, 2009;Minoshima & Hayashi, 2011).Some chaetotaxic characters of a putative third instar larva of E. (Holcophilydrus) niisatoi Minoshima were also briefly mentioned by Minoshima (2017).
Larval characters in Hydrophiloidea, including chaetotaxy, have proved to be a valuable tool for systematic and phylogenetic studies (e.g.Archangelsky, 2004;Fikáček, 2006;Fikáček et al., 2013;Minoshima et al., 2013;Archangelsky et al., 2021;Rodriguez et al., 2021).However, the limited number of chaetotaxic descriptions poses two difficulties: 1-limits our understanding of intrageneric variation; 2-it also limits the use of this source of characters in phylogenetic research since most generic larval chaetotaxic diagnoses are based on just one or two species.I have gathered a significant amount of larval material of Enochrus species through the years.Part of this material has been described following a general morphological approach (Archangelsky, 1997(Archangelsky, , 1999(Archangelsky, , 2002)), but chaetotaxic descriptions of these species were never presented before since the chaetotaxic and morphometric systems were not developed at the time (Fikáček, 2006;Fikáček et al., 2008;Torres, 2008;Byttebier & Torres, 2009).The aim of this contribution is to increase larval knowledge of Hydrophilidae by describing in detail the chaetotaxy and morphometry of five species of Enochrus belonging to three subgenera: Hugoscottia, Lumetus and Methydrus.This information will be very useful to researchers working in hydrophiloid phylogeny using larval characters; additionally, this is the first chaetotaxic description of larvae of the subgenus Lumetus.
This paper focuses on the description of the chaetotaxic and morphometric characters of the head capsule and head appendages; for general morphological characters of these species the following descriptions should be consulted (Richmond, 1920;Wilson, 1923;Gundersen, 1967;Archangelsky, 1997Archangelsky, , 2002)).

Methods.
Adults of all studied species were reared in laboratory, for most species the complete life cycle was obtained; for E. (M.) ochraceus only first and third instar larvae were available and for E. (L.) hamiltoni only third instar larvae were available.Larval specimens were cleared in warm lactic acid, dissected and mounted on glass slides with Hoyer's medium.Observations (up to 1000×), photographs and drawings were made with a Leica DMLB compound microscope with a camera lucida and a Leica S6D dissecting microscope with a photographic camera attached.Drawings were scanned and digitally edited, photographs were assembled using the freeware program CombineZP (Hadley, 2010).
Morphometry.Diverse measurements of the head capsule and head appendages were taken with a micrometer and were used to calculate ratios.Measured structures were adjusted as parallel as possible to the plane of the objective.The following measurements were taken; TL: total body length, measured in dorsal view, from anterior margin of frontoclypeus to posterior margin

MATERIAL AND METHODS
of the median lobe of spiracular atrium; MW: maximum body width, measured at level of prothorax; HL: head length, medially along epicranial stem from anterior margin of frontoclypeus to occipital foramen; HW: maximum head width; AL: length of antenna, derived by adding the lengths of the first (A1L), second (A2L) and third (A3L) antennomeres; SeL: length of antennal sensorium; SL: length of stipes; MPL: length of maxillary palpus, obtained by adding the lengths of the first (MP1L), second (MP2L), third (MP3L) and fourth (MP4L) palpomeres; ML: length of maxilla, derived by adding SL and MPL, cardo omitted; LPL: length of labial palpus, obtained by adding the lengths of the first (LP1L) and second (LP2L) palpomeres; LigL: length of ligula; MtW: maximum width of mentum; PrmtL: length of prementum, measured medially on dorsal side; PrmtW: maximum width of prementum.
Chaetotaxy.Primary (present in first-instar larva) and secondary (arising in later instars) setae and pores were identified in the cephalic capsule and head appendages.Primary sensilla were labeled by comparison with the ground plan of chaetotaxy of Hydrophilidae (Fikáček et al., 2008;Byttebier & Torres, 2009).Homologies were established using the criterion of similarity of position (Wiley, 1981).Sensilla are coded with a number and two capital letters, usually corresponding to the first two letters of the name of the structure on which they are located.Chaetotaxic descriptions of first and third larval instars are presented, second instar larvae are very similar to third instars.The following abbreviations were used.AN: antenna; FR: frontale; LA: labium; MN: mandible; MX: maxilla; PA: parietale; gAN: group of antennal sensilla; gAPP: group of sensilla on the inner appendage of the maxilla; gFR1, gFR2: group of sensilla on the frontale; gLA: group of sensilla on the labial palp; gMX1: group of sensilla on the maxillary palpomere 4; gMX2: group of sensilla on inner margin of maxillary stipes.

First instar larva
Head capsule (Fig. 2a-c): Frontale with 43 sensilla; seta FR1 moderately long, setae FR5 and FR6 subequal in length; gFR1 with six dorsal setae and one minute ventral seta below third seta from the right; first and second setae on right side shorter than remaining others; each gFR2 with two short setae and one small mesal pore.Parietale with setae PA7, PA12-14 and pore PA15 arranged in an almost transverse line; setae PA26, PA28 and pore PA27 not closely aggregated, PA27 closer to PA28 than to PA26; setae PA26 and PA16 at same distance from anterior margin of parietale.Antenna (Fig. 3a): Setae AN10-11 on membrane connecting A2 and A3.

Third instar larva
Head capsule (Fig. 4a-c): Frontale without secondary sensilla; right two setae of gFR1 proportionally larger than in first instar larvae.Parietale with several secondary sensilla distributed as follows: dorsally with four short setae between pore PA6 and seta PA7, one minute seta near PA8, one rather short seta between PA8 and PA9, one pore close to PA9, two rather short setae near PA13 and PA14; ventrally with one seta posterior to PA18 and on seta distal to PA16.Mandibles (Fig. 5b-c): Several secondary sensilla distributed as follows: one minute seta posterior to MN4, two minute setae posterior to MN1, a group of three short basal setae close to outer face and one pore placed more mesally than this group.
Maxilla (Fig. 5d-e): Outer face of stipes with two ventral secondary setae, one long mesal to MX5, the other short, halfway between pores MX2 and MX4.
Labium (Fig. 5f-g): Seta LA3 positioned more distally; mentum with nine or ten stout secondary setae dorsally, four on distal margin and two or three on each lateral face; ventrally with one short seta on each anterolateral corner.

Third instar larva
Head capsule (Fig. 6a-c): Frontale with 44 primary sensilla; seta FR1 moderately short, seta FR5 slightly shorter than FR6; gFR1 with six stout dorsal setae (specimen illustrated with one supernumerary seta, therefore bearing seven dorsal setae), first seta on right side shorter than remaining ones, ventrally with two minute setae between middle dorsal setae; each gFR2 with two minute setae and a small mesal pore; frontale with short secondary setae distal to FR1 (illustrated specimen with one on right side and two on left side).Parietale with numerous secondary sensilla distributed as follows: dorsally with seven to eight short setae between pore PA6 and seta PA7, one short seta near PA8, one rather long seta near PA9, one seta near PA13, one pore between seta PA9 and pore PA19, one short seta posterior to PA21; ventrally with four short setae between pore PA30 and seta PA16, one more seta distal to pore PA17.Mandibles (Fig. 7b-c): Seta MN1 rather short; seta MN5 closer to apex of mandible than to MN4; one minute secondary seta between MN1 and MN4, five short secondary setae posterior to MN1 two closer to MN1, remaining three more basal.
Maxilla (Fig. 7d-e): Outer face of stipes with two ventral secondary setae, one long mesal to MX5, the other shorter, distal to pore MX2.
Labium (Fig. 7f-g): Dorsal surface of mentum with 14 stout secondary setae, four on distal margin and five on each lateral face; ventrally with one short seta on each anterolateral corner.

Third instar larva
Head capsule (Fig. 10a-c): Frontale lacking secondary sensilla.Parietale with several secondary sensilla distributed as follows: dorsally with two to four rather short setae between pore PA6 and seta PA7, one very short seta mesal to PA8, another short seta between PA8 and PA9, one pore close to PA9, one minute seta behind PA21, one short seta near PA13 and another posterior to pore PA15; ventrally with two setae between PA16 and PA18 and two short setae distal to PA16 and PA17.Mandibles (Fig. 11b-c): Several secondary sensilla distributed as follows: one minute seta between MN4 and MN1, two minute setae posterior to MN1, a group of four short basal setae close to outer face.Maxilla (Fig. 11d-e): Outer face of stipes with two secondary setae, one long mesal to pore MX4, the other short, near pore MX2; setae MX16 and MX23 proportionally shorter.
Labium (Fig. 11f-g): Mentum with eight or nine stout secondary setae dorsally, three or four on distal margin and two or three on each lateral face; ventrally with one short and slender seta on each anterolateral corner.
Labium (Fig. 13f-g): Distance between pores LA7 subequal or less than that between setae LA6; distance between pores PA8 slightly larger than that between setalike sensilla LA9; pores LA11 at base of ligula; setae LA10 on membrane below ligula.

Third instar larva
Head capsule (Fig. 14a-c): Frontale lacking secondary sensilla.Parietale with several secondary sensilla distributed as follows: dorsally with one to three short setae between pore PA6 and seta PA7, one very short seta mesal to PA8, another long seta between PA8 and PA9, one pore close to PA9, one minute seta behind PA21, one short seta mesal to PA13 and another laterad to PA13; one short seta posterior to pore PA15; ventrally with one short seta between distal to PA18, two short setae distal to PA16 and PA17.Antenna (Fig. 15a): Antennal sensorium (SE1) proportionally shorter than A3.
Mandibles (Fig. 15b-c): Several secondary sensilla distributed as follows: one minute seta between MN4 and MN1, one or two very short setae posterior to MN1, a group of four short basal setae close to outer face.Maxilla (Fig. 15d-e): Stipes with two secondary setae, one long mesal to pore MX4, the other short, near pore MX2 on outer face; setae MX16 and MX23 proportionally shorter.
Labium (Fig. 15f-g): Mentum with nine or ten stout secondary setae dorsally, four on distal margin and two or three on each lateral face; ventrally with one short and slender seta on each anterolateral corner.

Third instar larva
Head capsule (Fig. 18a-c): Frontale lacking secondary sensilla, specimen illustrated missing right seta FR8.Parietale with several secondary sensilla distributed as follows: dorsally with three short setae between pore PA6 and seta PA7, one very short seta mesal to PA8, another long seta between PA8 and PA9, one pore close to PA9, one short seta mesal to PA13 and one short seta posterior to pore PA15; ventrally with one short seta between distal to PA18, two short setae distal to PA16 and PA17.Antenna (Fig. 19a): Antennal sensorium (SE1) proportionally shorter than A3.
Mandibles (Fig. 19b-c): Several secondary sensilla distributed as follows: one minute seta between MN4 and MN1, one very short seta posterior to MN1, a group of three short basal setae close to outer face.Maxilla (Fig. 19d-e): Stipes with two long secondary setae, one mesal to pore MX4, the other near pore MX2 on outer face; seta MX16 proportionally shorter, seta MX24 proportionally longer.
Labium (Fig. 19f-g): Mentum with twelve stout secondary setae dorsally, four on distal margin and four on each lateral face; ventrally with one short and slender seta on each anterolateral corner.

Comparative notes among subgenera
A comparative study of the chaetotaxy among known species of Enochrus provides an important number of characters useful to distinguish among subgenera.Table II summarizes all chaetotaxic characters useful to tell apart larvae of the subgenera Holcophilydrus, Hugoscottia, Lumetus and Methydrus; the chaetotaxy of the subgenera Enochrus and Hydatotrephis remains unknown.
The shape of the nasale, which bears a number of small irregular teeth, was used to distinguish between species by different authors (Richmond, 1920;Wilson, 1923;Böving & Henriksen, 1938;Fernández, 1992;Archangelsky, 2002;Watts, 2002;Byttebier & Torres, 2009).However, the number of teeth on the nasale does not seem useful when comparing larvae of several species for the following reasons: in first place there is some overlap in the number of teeth among species, for example both E. (H.) variegatus and E. (M.) vulgaris have 6-7 teeth while E. (H.) tremolerasi has 6 (Archangelsky, 2002;Byttebier & Torres, 2009); secondly in some species the nasale does not have a definite number of teeth and appears as irregularly serrated (e.g.E. hamiltoni, Fig. 6c); and finally in many specimens the nasale is worn out and it is difficult to count the teeth.
A special paragraph addressing the minute ventral setae of gFR1 should be made here.Two ventral minute setae in gFR1 were mentioned for E. (Hugoscottia) variegatus by Byttebier & Torres (2009) and after this mention they were not recorded for other species until now.Enochrus (Hugoscottia) tremolerasi has apparently only one (Figs.2c and 4c), below the third dorsal tooth of gFR1 from the right; this seta was found in all instars.Enochrus (Lumetus) hamiltoni has two ventral setae on gFR1 (Fig. 6c).Minoshima (pers. com.) confirmed that larvae of E. (Holcophilydrus) simulans have at least one minute ventral seta on gFR1, and that larvae of E. (Methydrus) japonicus have two minute ventral setae.I have not been able to find them in the three Methydrus larvae described in this paper.Apparently due to the small size of these ventral setae and the irregular shape of the serrated nasale of Enochrus larvae, these setae are very difficult to visualize, therefore their presence cannot be ruled out.

Variation within subgenera
Larvae within the same subgenus are very similar to each other, nevertheless some characters show variability (Table II).One interesting finding is that larvae of E. (Methydrus) japonicus display several differences with DISCUSSION other known Methydrus larvae.The frontale in third instar larvae of E. (M.) japonicus has one or two secondary setae distal to FR1 (Fig. 25a in Minoshima & Hayashi, 2011); all other known third instars of Methydrus lack secondary setae on the frontale.Additionally, gFR1 of E. (M.) japonicus has two minute ventral setae (Minoshima, pers. com.), these are apparently absent in other known Methydrus species.Pore PA3 in E. (M.) japonicus, is posterior to seta PA4, while in the remaining Methydrus species pore PA3 is located laterally to seta PA4.Third instar larvae of E. (M.) japonicus have secondary setae on the inner margin of the stipes, therefore conforming a distinctive gMX2 (Minoshima & Hayashi, 2011); these secondary setae are absent in all other known third instar Methydrus larvae.Secondary setae on the inner margin of the stipes (gMX2) are also found in third instar larvae of Enochrus (Lumetus) hamiltoni and E. (Holcophilydrus) satoi (Minoshima, 2017)

Possible diagnostic characters of Enochrus and Enochrinae
One interesting character that all described species of Enochrus share is the asymmetry in the distribution of sensilla FR11-13, already mentioned by Minoshima & Hayashi (2011).On the right side pore FR13 is located between pore FR11 and seta FR12 (e.g.Figs.2c, 6c), whereas on the left side seta FR12 is positioned between pores FR11 and FR13 (e.g.Figs.2c, 6c).This character seems unique within Enochrinae since it is absent in the other known larvae of this subfamily, Cymbiodyta and Notionotus (Perkins, 1972;Archangelsky, 1997;Fikáček, 2006;pers. obs.).
Another character that seems common to most described Enochrus larvae is the reduction in size of some of the right setae in gFR1 (e.g.Figs. 2c,6c,8c).In Enochrus (Hugoscottia) the first two setae of the right are smaller than the remaining ones; in E. (Lumetus) hamiltoni, the first seta on the right is smaller; in Enochrus (Methydrus) the second seta on the right is smaller.Apparently, Cymbiodyta and Notionotus show no reduction of setae on the right side of gFR1 (Perkins, 1972;Archangelsky, 1997;Fikáček, 2006;pers. obs.).
A character present in all known second and third instar larvae of Enochrus is a short ventral seta on each anterior corner of the mentum (e.g.Figs.5g, 7g); this seta is also present in second and third instars of Cymbiodyta (pers.obs.); since Notionotus second and third instar larvae are unknown this character could not be evaluated for this genus.Finally, first instar larvae of all known Enochrus species bear a few small spicules on the ventral side of the second antennomere (Byttebier & Torres, 2009;Minoshima pers. com.).These spines are also present in first instars of Cymbiodyta and Notionotus larvae (Perkins, 1972;pers. obs.).
Even though no wide phylogenetic analysis of Enochrinae using molecular and/or adult characters has been performed, there is some evidence that the relationships within Enochrus are not completely resolved CONCLUSIONS (see Fig. 3, Short & Fikáček, 2013).Enochrus larvae share several diagnostic characters that easily distinguish them from other genera within Enochrinae.Moreover, despite the great similarity they show, several morphological and chaetotaxic characters are useful to discriminate among larvae of the different subgenera.Regardless of this, some characters show variation within the same subgenus; this illustrates how important is the description of new larvae within genera that are supposed to be considerable well known (Rodriguez, 2021).
This study shows that Enochrus larvae display a great similarity in the chaetotaxy among species of the same subgenus, making it difficult to tell them apart; nevertheless, some species (e.g.E. (M.) japonicus) are quite distinctive within the subgenus and are easy to differentiate, therefore it is necessary to increase the number of larval descriptions in order to better understand this fact.
Description of the chaetotaxy and morphometry of larvae of the subgenera Enochrus (s.str.) and E. (Hydatotrephis) should be a priority with the aim to complete the larval knowledge of the genus.Knowledge of these larvae will give us a more complete picture of larval characters and will provide invaluable information for future phylogenetic analyses.
CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) is acknowledged for supporting systematic research.I am indebted to Yȗsuke Minoshima and Patricia L. M. Torres for their help checking some chaetotaxic characters in Enochrus larvae.The comments and suggestions of two anonymous reviewers and the Editors improved this contribution and are also acknowledged.

Table II . Chaetotaxic and morphometric characters useful to distinguish among larvae of known subgenera of Enochrus.
. Within Holcophilydrus, only third instar larvae of E. (H.) satoi have gMX2 on the stipes; third instars of two other known species, E. (H.) simulans and E. (H.) umbratus lack this character.