Phylogenetic relationships and larval morphology of the recently described diving beetle genus Laccomimus (Coleoptera: Dytiscidae: Laccophilinae)

The larvae of the diving beetle genus Laccomimus Toledo & Michat, 2015 (Laccophilinae) are studied for the first time, based on detailed descriptions and illustrations of all instars of L. distinctus Toledo & Michat, 2015, with particular emphasis on morphometry and chaetotaxy. The phylogenetic relationships of this genus within the tribe Laccophilini are analyzed cladistically. Laccomimus is recovered as part of a clade that includes the genera Australphilus Watts, 1978, Neptosternus Sharp, 1882 and Laccophilus Leach, 1815 and is characterized by the presence of natatory setae on the tibia and tarsus, and within this clade it is a sister to the other genera. Third-instar larvae of Laccomimus and Africophilus Guignot, 1948 share the mediodistal insertion of the seta CO7 on the mesoand metacoxa, a short and spine-like seta TI6 on the metatibia, a ventrally sclerotized abdominal segment V and a short urogomphus. These characters are considered plesiomorphic and indicate a basal position of both genera within the Laccophilini. All instars of Laccomimus are characterized by the anterolateral lobes of the frontoclypeus clearly projecting beyond the anterior margin and the last abdominal segment strongly elongated. Diagnostic features of the first instar are: frontoclypeus unmodified posteriorly, lamellae clypeales thin and hair-like, pores ANe, MXb–d–f–i, LAb–c and seta TR3 absent, seta LA3 and an additional dorsal pore present on the prementum, abdominal tergites I–VII with anterior transverse carina, and ventral surface of the abdominal segment VI sclerotized.


IntroductIon
On a collecting trip in the Humid Chaco eco-region (Chaco Province, Argentina) seven years ago the senior author collected some unusual and particularly interesting diving beetle larvae in a pond with vegetation.After a more detailed examination under a microscope these larvae were thought to belong to the subfamily Laccophilinae.The resemblance with the (so far) only laccophiline genus known from Argentina (Laccophilus Leach, 1815) was, however, rather vague.The occurrence in the same habitat of adults of a small laccophiline species apparently not belonging to Laccophilus raised the question whether these unusual larvae actually belong to another genus, with Laccodytes Régimbart, 1895 being the most likely candidate.The adults collected in that habitat were recently studied in detail as part of an extensive revision of some Laccophilini genera (Toledo et al., 2010;Toledo & Michat, 2015), which confirmed they do not belong to Laccophilus or Laccodytes but to a new genus, Laccomimus Toledo & Michat, 2015.
The genus Laccomimus includes 12 species of small diving beetles (less than 3 mm in length) widespread in tropical America from Florida in the US to central Argentina, most commonly found in lentic or stagnant water rich in debris and vegetation (Toledo & Michat, 2015).In particular, the adult specimens collected along with the larvae were described as a new species, L. distinctus Toledo & Michat, 2015, and placed in an isolated phylogenetic position as the sister group of all the other species in this genus (Toledo & Michat, 2015).All these findings make the remarkable larvae collected years ago a very interesting tar-get for study, as the immatures of Laccomimus have been unknown.
On the basis of adult characters, Laccomimus was found to be reliably placed within the tribe Laccophilini.After a cladistic analysis, it was hypothesized that it is a sister group of the Oriental genus Laccosternus Brancucci, 1983, both taxa forming a clade sister to Laccophilus (Toledo & Michat, 2015).Larval morphology of the subfamily Laccophilinae is imperfectly known, with the larvae of several genera still unknown.Larval characters, however, have proven to be very useful in the study of the phylogenetic relationships within Dytiscidae.As different expressions of the same genotype, larval characters help to complement adult characters that are traditionally the primary basis for classification.In particular, larval chaetotaxy is a significant source of characters both for diagnosis of the genera and species and for the study of the phylogenetic relationships within the Laccophilinae (Alarie et al., 2000, 2002a;Michat, 2008).The development of a system of nomenclature for the primary sensilla (setae and pores) of first-instar larvae of this subfamily (Alarie et al., 2000) has revealed the taxonomic and phylogenetic value of this character set.
This paper is a contribution to a better understanding of the larval morphology of the Laccophilinae and has the following goals: (1) to describe and illustrate in detail the three larval instars of L. distinctus, with particular emphasis on an analysis of chaetotaxy of the cephalic capsule, head appendages, legs, last abdominal segment and urogomphi; (2) to compare the larval characters of Laccomimus with those of other laccophiline genera for which the  , 2000).Particular emphasis was placed on including characters of the third instar because this is the only instar known for all species.The lack of information on the first instar of several species, which results in many question marks in the matrix, prevented the inclusion of most characters from this semaphoront.All characters were treated as unordered and equally weighted.Multistate characters were treated as nonadditive.An exact solution algorithm (implicit enumeration) was implemented to find the most parsimonious trees.Bremer support values were calculated using the commands "hold 20000", "sub n" and "bsupport", where "n" is the number of extra steps allowed.The process was repeated increasing the length of the suboptimal cladograms by one step, until all Bremer values were obtained (Kitching et al., 1998).Bootstrap values were calculated using the following parameters: "standard (sample with replacement)"; 2,000 replicates.Instar I (Figs 1-15).Colour.Larva entirely brown except head appendages and legs, which are light brown, and the testaceous membranous parts.
Body (Fig. 1).Subcylindrical, narrowing towards abdominal apex.Measurements and ratios that characterize the body shape are shown in Table 1.
Head.Cephalic capsule (Figs 2-3).Subovate, somewhat longer than broad; surface covered with reticulated microsculpture; maximum width at level of stemmata; without neck constriction; occipital suture absent; ecdysial line well marked, coronal line moderately long; occipital foramen broadly emarginate ventrally; posterior tentorial pits visible ventrally; FR subtriangular, with two blunt egg bursters at base, anterior margin rounded medially, anterolateral lobes rounded, somewhat projecting beyond an-larvae are described; and (3) to study the phylogenetic relationships of this genus within the tribe Laccophilini based on larval characters.

Preparation and description of the larvae
Six specimens of instar I, five of instar II and seven of instar III were used for the descriptions.Larvae were collected in association with adults at the following locality: Argentina, Chaco Province, El Cachapé refuge, 4.xii.2008,small semi-permanent pond about 15 m long, 5 m wide and 70 cm in depth, with vegetation growing around the margins (see Figs 131 and 132 in Toledo & Michat, 2015).It is most likely these larvae belong to L. distinctus as only adults of this species of Laccomimus were collected at this site.
Larvae were cleared in lactic acid, dissected and mounted on glass slides in polyvinyl-lacto-glycerol.Microscopic examination at magnifications of up to 1,000× and drawings were made using an Olympus CX31 compound microscope equipped with a camera lucida.Drawings were scanned and digitally inked using a Genius PenSketch tablet.Voucher specimens are deposited in the collection of M.C.Michat (Laboratory of Entomology, Buenos Aires University, Argentina).
The methods and terms used in the present paper follow those employed in previous papers dealing with the larval morphology and chaetotaxy of members of Laccophilinae.The reader is referred to Alarie et al. (2000, 2002a) and Michat (2008) for a complete list and additional explanations of the terms used in the present study.

chaetotaxic analysis
Primary (present in instar I) and secondary (added throughout the ontogenetic development) setae and pores on the cephalic capsule, head appendages, legs, last abdominal segment and urogomphus were recorded.Sensilla were coded using two capital letters, in most cases corresponding to the first two letters of the name of the structure on which they are located, and a number (setae) or a lower case letter (pores).The following abbreviations were used: AB -abdominal segment VIII; AN -antenna; COcoxa; FE -femur; FR -frontoclypeus; LA -labium; MN -mandible; MX -maxilla; PA -parietal; PT -pretarsus; TA -tarsus; TI -tibia; TR -trochanter; UR -urogomphus.Setae and pores present in first-instar larvae were labelled using the ground plan of chaetotaxy of the subfamily Laccophilinae (Alarie et al., 2000(Alarie et al., , 2002a)).Homologies were determined by using the criterion of similarity of position (Wiley, 1981).Setae located on the apices of the maxillary and labial palpi were extremely difficult to distinguish due to their position and small size, and, therefore, they are not well represented in the drawings.
Chaetotaxy (Figs 1-15).Similar to that of a generalized Laccophilinae larva (Alarie et al., 2000, 2002a; Michat, 2008) except for the following features: Pore PAc absent; pores ANe and ANh absent; setae MX5, MX6 and MX9 absent; pores MXb, MXd, MXf and MXi absent; setae LA10 and LA12 absent; pores LAb and LAc absent; prementum with one additional pore on dorsal surface (near seta LA8); seta CO7 inserted more proximally on L1; seta TR3 absent; seta FE4 inserted somewhat more proximally; seta FE5 short, spine-like; FE with one additional spine-like seta on anteroventral surface; seta TI4 on L2 and L3 long, hair-like, inserted distally, on L1 short, spine-like, inserted more proximally; seta TI6 short, spine-like; seta TA1 on L1 and L2 short, spine-like, inserted distally, on L3 long, hairlike, inserted more proximally; pores of the couples TAc-TAd and TAe-TAf difficult to distinguish, there appears to be only one pore on each side; however, their presence in instars II and III indicates that they may be present also in instar I; setae AB2, AB9, AB10 and AB15 inserted more proximally; insertion of setae UR2 and UR3 contiguous; seta UR7 very long, inserted terminally on U1; although we were unable to distinguish seta UR1 on the urogomphus, we think it is present but obscured by the presence of basal spinulae (it is present in instars II and III).
Instar II (Figs 16-25).As instar I except for the following features: Table 1.Measurements of structures and their ratios for the three larval instars of Laccomimus distinctus.
Chaetotaxy (Figs 16-25).Cephalic capsule with numerous hair-like secondary setae, 3-4 spine-like setae on each lateral margin of PA, one spine-like seta on each side of dorsolateral surface of PA, and 2-4 spine-like setae on each side of ventrolateral surface of PA (Figs 17, 18); anterior margin of FR with nine short spine-like lamellae clypeales (Fig. 17); MN with one hair-like secondary seta on basoexternal margin (Fig. 19); prementum with one secondary pore on ventral surface (near seta LA1) (Fig. 20); thoracic tergites with secondary setae (Fig. 16), the marginal ones inserted on the apices of stout spinulae; secondary leg setation detailed in Table 2 and Figs 21 and 22; TR with one secondary pore on proximal portion; TI with one proximal natatory seta on posterodorsal margin; proTA without natatory setae; mesoTA with two natatory setae on posterodorsal margin; metaTA with four natatory setae on posterodorsal margin; abdominal sclerites with secondary setae (Fig. 16), the marginal ones inserted on the apices of stout spinulae; secondary setation on LAS detailed in Figs 23 and 24.
Table 2. Number and position of secondary setae on the legs of larvae of Laccomimus distinctus.Numbers separated by slash marks refer to pro-, meso-and metathoracic legs, respectively.A -anterior, D -dorsal, NS -natatory setae, P -posterior, Prproximal, V -ventral, Total -total number of secondary setae on the structure, excluding primary (ancestral and additional) and natatory setae.

Instar III (Figs 26-29). As instar II except for the following features:
Body.Measurements and ratios are shown in Table 1.Thorax.Non-functional spiracles present on mesothorax.Abdomen.Segment II with a single large ventral sclerite independent of dorsal sclerite; non-functional spiracles present on segments I-VII, each on the apex of a small lobe.Urogomphus (Fig. 29).U2 shorter than U1.

Chaetotaxy (Figs 26-29)
. Secondary setation on cephalic capsule (Fig. 26), thoracic and abdominal sclerites more abundant; anterior margin of FR with 12 short spine-like lamellae clypeales (Fig. 26); PA with 7-10 spine-like setae on each lateral margin, 3-4 spine-like setae on each side of dorsal surface, and 9-10 spine-like setae on each side of ventral surface (Fig. 26); secondary leg setation detailed in Table 2 and Figs 27 and 28; secondary setation on LAS detailed in Fig. 29.31).In the last years of their lives, Young and Spangler no longer worked on taxa now included in Laccomimus, and therefore this description was never published.The comparison with L. distinctus reveals that the mature larvae of both species are very similar.Below we give an extract of Young's description of L. bordoni, including only the characters that differ from those of L. distinctus.
Colour: dorsal surface of head dark brown except markings around ocular area, bases of antennae, narrow area along frontoclypeal margins and extreme base of head that are testaceous; ventral surface of head dark brown except for the testaceous oral appendages; antenna and mandible testaceous; pronotal sclerite dark brown except for the testaceous anterolateral angles, longitudinal stripe between median ecdysial cleavage line and lateral margins and the fine ecdysial cleavage line; mesonotal sclerite dark brown except for the testaceous anterolateral margins; metanotal sclerite same as mesonotal sclerite except for a smaller and less obvious testaceous area; abdominal sclerites dark brown, apices of sclerites VI-VIII testaceous; ventral sclerites dark brown; legs testaceous; membranous areas creamy white.
Body: TL = 3.60 mm; MW = 0.85 mm.Abdomen: segment II with two large ventral sclerites independent of dorsal sclerite; terga of abdominal segments I-VI each with a lateral spiracle.
As we have not examined the larvae of L. bordoni, the morphological differences mentioned above should be approached with caution.The different degree of sclerotization on the ventral surface of abdominal segment II may be due to the different age of the larvae, and a more extensive sampling is needed to confirm this character.On the other hand, the presence of spiracles on the seventh abdominal segment is a common feature of Dytiscidae (including L. distinctus), therefore these structures may have been overlooked in L. bordoni.

character analysis
In total, 35 characters derived from the larval morphology and chaetotaxy were included, of which 34 were coded as binary and one as multistate (Appendix 1).The analysis of the data matrix (Appendix 2) with TNT resulted in three most parsimonious cladograms of 44 steps (CI = 0.81; RI = 0.88), which differed in the relative positions of the species of Laccophilus.The strict consensus was calculated (Fig. 32), in which Laccomimus is resolved as part of a large and weakly supported clade including also the genera Australphilus Watts, Neptosternus Sharp and Laccophilus.Within this group, Laccomimus is sister to the other genera, which form a well-supported clade.Character state changes are mapped on one of the most parsimonious trees (Fig. 33).

dIscussIon
Larval morphology of members of the subfamily Laccophilinae is still imperfectly known.Several genera are unknown as larvae and only third-instar larvae are known for others.This hampers considerably any attempt to study the phylogeny within the subfamily based on larval characters.In particular, the lack of knowledge on the first instar (only three out of 13 genera known as larvae) prevented the inclusion of most characters from this semaphoront.Therefore, we elected to perform the cladistic analysis based mainly on characters of the third instar, avoiding the introduction in the data matrix of an excessively high number of question marks.Based on these premises, the phylogenetic analysis provided here should be considered as preliminary and subject to changes when more genera and additional larval stages are described.
Our phylogenetic analysis supports the placement of Laccomimus within the tribe Laccophilini.All the genera of this tribe are characterized by the presence of twosegmented urogomphi (character 32.1) and of secondary setae on the pro-and mesotibia (character 21.1) and on the dorsal surface of the protarsus (character 23.1), all character states absent in Agabetes (Agabetini) (Fig. 33).The phylogenetic signal of these characters, however, is weak as long as the presence of two-segmented urogomphi is a common feature of Dytiscidae as found in the Hydroporinae (Alarie & Harper, 1990), most Agabinae (Alarie, 1995) and Copelatus Erichson, 1832 (Michat & Torres, 2009), and secondary setae on the pro-and mesotibia and dorsal  Within the Laccophilini, Laccomimus is part of a large clade including all the genera known as larvae except Africophilus Guignot, 1948 (Fig. 32), which is resolved as a sister to the rest of Laccophilini, in accordance with Alarie et al. (2000).The clade formed by all the genera except Africophilus is characterized by the presence of natatory setae on the posterodorsal surface of the tibia (character 22.1) and tarsus (character 24.1), which are absent in Africophilus and the outgroup (Agabetes) (Fig. 33).As with the previous characters, however, natatory setae on the posterodorsal surface of the tibia and tarsus are common features, present in several other subfamilies of Dytiscidae (see references above), and therefore cannot be used as strong evidence in support of the grouping mentioned above.The absence of natatory setae on the legs in Africophilus and Agabetes likely represents independent secondary losses associated with their peculiar habitats; Africophilus is hygropetric (Omer-Cooper, 1965), Agabetes inhabits the leafy substrate in woodland ponds (Spangler & Gordon 1973) and some specimens were even taken from a wet leaf pack well above the water line (Larson et al. 2000).
It is interesting to note that the specimen recorded as "Laccodytes sp1" in Ribera et al. ( 2008) is actually a species of Laccomimus (Ignacio Ribera, pers.comm.).If this is taken into account, the results of our phylogenetic analysis are in very good agreement with the molecular results of Ribera et al. (2008).In this later paper Laccomimus is resolved as a sister to Africophilus and both a sister to a clade that includes all the other genera of Laccophilinae studied, among which are Australphilus, Neptosternus and Laccophilus.Our results recover Laccomimus and Africophilus as paraphyletic with respect to the other genera, although this is apparently due to the absence of natatory setae on the legs of both Africophilus and the outgroup Agabetes, which is most likely an independent loss.Laccomimus and Africophilus share a number of larval characters considered to be symplesiomorphies (see below), which means that the paraphyly of the two genera could change if more characters are added.This is also reflected in the low support values (Fig. 32).
The next branch of the tree separates the genus Laccomimus from the remaining genera (Australphilus, Neptosternus and Laccophilus) (Fig. 32).This relationship is relatively well supported by several characters.In fact, the clade Australphilus + Neptosternus + Laccophilus is characterized by four synapomorphies: (1) seta CO7 inserted proximally on the meso-and metacoxa (character 14.1); (2) seta TI6 long and hair-like on metatibia (character 20.0); (3) ventral surface of abdominal segment V membranous (character 26.0); (4) urogomphus elongate (character 31.1) (Fig. 33).The branching pattern Laccophi-lus (Australphilus, Neptosternus) is well supported in our tree (Fig. 32) and is in accordance with previous results of Alarie et al. (2000, 2002a), demon strating that these three genera form a solid unit based on larval characters.Ribera et al. (2008), however, obtained a different branching pattern based on molecular data and a more comprehensive sampling of taxa.On the other hand, Laccomimus is supported by the anterolateral lobes of the frontoclypeus projecting beyond the anterior margin (character 2.1), which is shared with Agabetes but unique within the Laccophilini genera studied.This genus is also characterized by the apically rounded shape of the egg bursters (character 3.1), but this is a character of the first instar and therefore could not be evaluated for all the genera.
In a recent phylogenetic analysis of the Laccophilini based on adult characters (Toledo & Michat, 2015), Laccomimus is a sister group of Lacco sternus, and both taxa form a clade sister to Laccophilus.Unfortunately, the larvae of Laccosternus are unknown and therefore this hypothesis could not be tested in the present study.The relationship with Laccophilus, however, is rather distant in the larval tree and both genera differ in several larval characters (see Fig. 33).Laccomimus shares with Africophilus the mediodistal insertion of the seta CO7 on the meso-and metacoxa (character 14.0), a short and spine-like seta TI6 on the metatibia (character 20.1), the sclerotized ventral surface of abdominal segment V (character 26.1) and a short urogomphus (character 31.0).As similar states for these characters are present in the outgroup (Agabetes), they indicate a basal position of both genera within the Laccophilini.Interestingly, the frontoclypeus of the first instar is not modified in Laccomimus (character 1.0) (Fig. 2).A modified frontoclypeus, either truncate posteriorly as in Laccophilus or very narrow posteriorly as in Neptosternus, seems to be the rule among first-instar larvae of Laccophilini (De Marzo, 1976;Alarie et al., 2000;Michat, 2008).The fact that firstinstar larvae of Agabetes (Alarie et al., 2002a) also have an unmodified frontoclypeus is another argument in favour of a basal position of Laccomimus within the Laccophilini.It would be interesting to know if the frontoclypeus of Africophilus is modified, but unfortunately the first instar of this genus is unknown.
Including Laccomimus, the first instars of only three Laccophilini genera are described, which restricts the comparisons to a small fraction of the total number of genera.Nevertheless, it is interesting to discuss the most conspicuous differences exhibited by Laccomimus with respect to Laccophilus and Neptosternus (Alarie et al., 2000).It is well known that first-instar larvae of the Laccophilini have only two lamellae clypeales (De Marzo, 1976;Alarie et al., 2000;Michat, 2008), the lowest number recorded within the Dytiscidae.Laccomimus is not an exception, but unlike Laccophilus and Neptosternus, in which the lamellae are broad and spatulate, in this genus they are thin and hair like (Fig. 2) to the extent that they are difficult to distinguish from the other setae.Positional homology and presence of all other frontoclypeal setae commonly found in Dytiscidae, however, left little doubt that these two hair-like se-tae are in fact homologous to the lamellae clypeales.The pore MXd and the seta MX9 are absent on the maxilla of Laccomimus (Fig. 7), whereas they are present in Laccophilus and Neptosternus.Similarly, the labial pores LAb and LAc (Figs 9, 10) and the trochanteral seta TR3 (Fig. 11) are absent in Laccomimus and present in the other two genera.In contrast, the labial seta LA3 is present in Laccomimus, as well as an additional pore on the dorsal surface of the prementum (Fig. 9).These structures are absent in Laccophilus and Neptosternus.With respect to the abdomen, the tergites I-VII have an anterior transverse carina in Laccomimus, and the ventral surface of the segment VI is sclerotized, whereas segment VIII is considerably elongated as in the Cybistrini (Michat, 2006(Michat, , 2010; Alarie et al., 2011) (Fig. 1).In Laccophilus and Neptosternus, the tergites I -VII lack an anterior transverse carina, segment VI is membranous ventrally and segment VIII is very short.Finally, the first instar of Laccomimus is highly characteristic in that the pores ANe, MXb, MXf and MXi are absent (Figs 5, 7, 8).These pores are present not only in Laccophilus and Neptosternus but also in the first-instar larvae of all dytiscid genera currently known (except MXb, which is absent in Hydroporinae).

Fig. 32 .
Fig. 32.Strict consensus cladogram obtained from the cladistic analysis based on 12 terminal taxa of Laccophilinae, with Bremer support values indicated above branches and Bootstrap support values indicated below branches.