Larval morphology and phylogenetic position of Drusus balcanicus

In a recent 3-gene phylogeny of the trichopteran subfamily Drusinae Banks 1916, molecular data clearly correlated with the morphology and feeding ecology of larvae. The largest of three main groups, the Drusinae grazer clade, exhibits an unusual larval feeding ecology for Limnephilidae, and is the most diverse group. In this paper we describe four previously unknown Drusinae larvae included in this clade: Drusus balcanicus Kumanski, 1973 (micro-endemic to Eastern Balkans), Drusus botosaneanui Kumanski, 1968 (Dinaric Western Balkans, Hellenic and Eastern Balkan, Asia Minor), Drusus serbicus Marinković-Gospodnetić, 1971 (micro-endemic to Dinaric Western Balkans), and Drusus tenellus (Klapálek, 1898) (Carpathians, Dinaric Eastern Balkans). Characteristically, the larvae of these species have toothless mandibles typical of the Drusinae grazer clade. Larvae and adults were unambiguously associated using a phylogenetic analysis based on two mitochondrial [mtCOI, mtLSU (=16S) rDNA] and two nuclear genes (nuWG, nuCAD). In addition, information on the morphology of the larvae is given and the diagnostic features necessary for identification are illustrated.


Molecular study
We used phylogenetic analysis to associate the larvae.We inferred phylogenetic trees based on molecular sequence data from two nuclear and two mitochondrial genes of the four target species and those previously published for 43 other species of Drusinae (Supplementary table S1).We extracted DNA from larval and adult specimens using the DNEasy Blood & Tissue Kit (Qiagen) following the manufacturer's protocol.PCRs were carried out in 10 µl of solution.PCR procedures and primers are listed in Table 1.PCR products were sequenced on an ABI 3177XL capillary sequencer at the Biodiversity and Climate Research Laboratory Centre.Sequences were edited in Geneious vR7 (biomatters).Sequences were aligned using the Muscle-plugin in Geneious vR7.
We inferred phylogenetic trees for each locus separately using a Bayesian/MCMC analysis implemented in MrBayes v3.2.1 (Ronquist et al., 2012).Nucleotide substitution models were selected using the Bayesian Information Criterion in the model test module of MEGA v5.2 (Tamura et al., 2011).In the protein coding genes, nucleotide substitution models were identified separately for each codon position (see Table 2 for codon-specific selected models).We did not partition the 16SrDNA fragment.All model estimations were performed using all sites, i.e. including the gaps in LSU.The B/MCMC analysis was based on 2 parallel runs with six chains each that explored tree space for 10 million generations.Phylogenetic trees were based on 15,000 trees (2 × 7,500) following a 25% burn-in phase.We assessed the parameter files in Tracer Version 1. 4.6 (Drummond & Rambaut, 2007) to determine if each run had reached stationarity.We used the As caddisfly larvae are important indicator taxa for monitoring water quality (Barbour et al., 1999;Barbour & Yoder, 2000;AQEM consortium, 2002;Graf et al., 2002;Hering et al., 2006) and are frequently used as bioindicators (sensitive species) (Moog et al., 2002;Graf et al., 2002), the newly-described larvae will improve resolution of ecological assessment procedures.Further, larval morphology is also seen as an important tool in phyloge netics and taxonomy (van Emden, 1957;Meier & Lim, 2009;Minoshima et al., 2013).The descriptions of the four new Drusinae larvae will, therefore, also increase our present knowledge of the phylogenetic structure of the Drusinae grazer clade sensu Pauls et al. (2008).

Species collection
Adults and larvae of Drusus serbicus, D. botosaneanui, D. bal canicus, and D. tenellus were collected by hand on the Balkan Peninsula (for locations see Material examined).
The material intended for sequencing was placed in pure 96% alcohol and that for morphological analyses in pure 70% ethanol in order to keep the specimens more flexible.

Morphological study
Morphological terminology, including setal nomenclature, follows Wiggins (1998).The larvae were described in terms of the set of morphological characters for Drusinae defined by Waringer & Graf (2011).Larvae were studied and photographed using a Nikon SMZ 1500 binocular microscope with DS-Fi1 camera and NIS-elements D 3.1 image stacking software, which combine 8 to 42 frames in one focused image.The two 5th instar larvae of D. balcanicus and the three larvae of D. botosaneanui, D. serbi cus and D. tenellus are deposited in the collection of J. Waringer (Vienna, Austria).
For SEM microscopy, two fifth instar larvae of Drusus serbicus were air dried, gold coated using a BAL-TEC SCD 005 sputter coater and examined using a JEOL JSM-6390lv scanning electron microscope.
Comparative material of other Drusinae species included the following (all larvae preserved in pure 70% ethanol): Drusus franzressli Malicky, 1974 (two 5th instar larvae), D. spelaeus (Ulmer, 1920)  average standard deviation of split frequencies between runs after 2,500,000 generations if both runs reached the same optimality space.

Identification of the larvae
The putative conspecific larvae always clustered in monospecific clades with adults (Table 3, supplementary Figs S2a-d).There are, however, some weaknesses in the resolution of the larval association clades.In the WG phylogeny, clades including D. botosaneanui and D. bal canicus were not significantly supported (pp < 0.95).For COI and CAD all sequences of D. balcanicus are basal to a highly supported clade for D. discophoroides Kumanski, 1979 (pp = 1.0), but are not grouped in supported clades.For LSU there is a similar situation regarding D. tenellus.However, these topological inconsistencies only insignificantly weaken the overall associations of adults with larvae, which are further supported by identical haplotypes in all species except D. tenellus.
Head.Head capsule coarsely granulated, almost circular in shape and hypognathous (Figs 1A-C), dorsally with blackish muscle attachment spots.Ventral parietalia sections, submentum, maxillolabial sclerites and premandibular areas medium to orange brown (Figs 1C, D).Yellowishwhite ring around each eye (Fig. 1C).In lateral view, head capsule with carina (0.40-0.45 mm long and approximately 0.04 mm wide) starting a short distance from anterior margin of eye and extending to frontomedian corner of frontoclypeus (Fig. 1C, arrow).

Drusus klapaleki
Antennae arise on dorsal rim of lateral carina and halfway between eye and anterior head margin (Fig. 1E, arrow), each consisting of 1 short cylindrical base and 1 short flagellum.On each parietale there are 10 dorsal and 2 ventral primary setae, with primary setae 5, 9 and 14 long and conspicuous (Figs 1B, C, E).Six primary setae on each side of frontoclypeus, 3 of them along anterior border.Labrum medium to light brown, with setal brush and primary setae 1-3 on anterolateral margins; on dorsal area, setation consists of primary setae 4-6 (Figs 1A, E).
Ventral apotome elongated triangular, medium to light brown, postgenal suture approximately 55% of apotome length (Fig. 1D).Blackish brown to dark brown mandibles lacking terminal teeth along edges as well as lacking ridges in central concavity (Figs 1D, E).
Metanotum partially covered by 3 pairs of medium to dark brown sclerites.Anterior metanotal sclerites (sa1) table 3. Results of larval associations based on phylogenetic reconstruction for each of the four loci.The 5th instar larvae of the target species are deposited in the collection of J. Waringer (Vienna, Austria).m -males, f -females, l -larvae, pp -posterior probability.very large, broadly ovoid, strongly tapering laterally, each with black anterior margin; separated by less than own length (Fig. 2E).Approximately 15 setae per sclerite (Fig. 2E).Row of setae present between small posteromedian sclerites (sa2); each sclerite bears 14-17 setae.Small setal group present between each lateral (sa3) and posteromedian sclerite (sa2); each sa3 with approximately 25-30 setae, concentrated anteriorly (Fig. 2E).Legs orange brown with   berance.Sharply delimited basal sclerites present in about 30% of these setae; without setal group posterior to dorsal protuberance (Fig. 2E).Lateral protuberances lacking pos-terior sclerites (Fig. 3C).A continuous band of anterolateral setae present in front of each lateral protuberance, linking each dorsal and ventral sa3 setal group (Fig. 3C).First   abdominal sternum with ventral setal areas sa1, sa2 and sa3 fused, creating continuous field of setae; basal sclerites of setae on the central area of the first abdominal sternum mostly small and inconspicuous except for four larger basal sclerites near midline and immediately ventral to the lateral protuberances.Basal sclerites never fuse with one another (Fig. 3D).Eighth abdominal dorsum bears two to four long posterodorsal setae (pds) (Fig. 3E, dotted oval).Only 1 posterolateral seta present on each half of 9th abdominal dorsum (Fig. 3E, arrows).
All gills single filaments.Dorsal gills present at most on the 2nd (presegmental position) to the 7th segment (presegmental position).Ventral gills on the 2nd (postsegmental) to 8th segment (presegmental).Lateral gills lacking.Lateral fringe extending from posterior third of 2nd to middle of 8th abdominal segment; in addition, a prominent seta surrounded by a small number of isolated lateral fringe setae on anterior border of 2nd segment.Light brown sclerite on 9th abdominal tergum semicircular (Fig. 3E); 7-8 long and several shorter setae present along its posterior border, 1-2 of the long setae take the position of central intermediate c setae (Fig. 3E).Anal prolegs of limnephilid type, light to medium brown, with light muscle attachment spots.Anal claws medium brown, each with 1 small accessory hook (Fig. 3F).
Habitat.This species inhabits the epirhithral section of oxygen-rich streams with high to moderate currents, but is also encountered near the source (hypocrenal region) down to the metarhithral zone.Drusus serbicus is a grazer feeding on epilithic biofilms and associated algae.

Key to larvae of species of Drusus of the grazer clade having spinule areas on head capsules
As in the other species in the Drusinae grazer clade, the mandibles are spoon-shaped (lack terminal teeth and ridges in central cavity; Fig. 1E).The larva of Drusus ser bicus is similar to six Drusinae species from the Balkan Peninsula, which have a small field of spinules (= small spines approximately 0.03 mm long) posterior to their eyes ( Figs 1E, F).Based on the recent detailed descriptions of Kučinić et al. (2008Kučinić et al. ( , 2010Kučinić et al. ( , 2011a, b, in press) , b, in press) D).Labrum dark brown, with setal brush (Fig. 4D).Ventral apotome yellowish to light brown and with postgenal suture approximately 70-75% of apotome length.Head capsule lacking any additional spines, bristles or areas of spinules.
Thorax.Pronotum with adjacent series of granuli creating ribbed structures (Fig. 4F).Dorsal profile in lateral view with posterior half of pronotum rounded, this curvature creates a distinct step leading down to anterior, lower part of pronotum (Fig. 4F).Lateral ridge lacking.In total, 35-40 dark setae of varying lengths distributed over each pronotal half.Prosternite very light and indistinct.
Abdomen.Centre of 1st abdominal sternum with large or medium concentrations of fused basal sclerites of setae, creating multilobed patterns of sclerotized areas positioned mostly posterior of the two largest basal sclerites (Figs 5B-D).
Case. Larval case 10.6-10.7 mm long (n = 2), curved, slightly conical.Width at anterior opening 2.5-2.6 mm and at posterior opening 1.8-1.9mm, consisting of mineral particles (sand grains of mixed size; Fig. 5E).Habitat.Drusus balcanicus is confined to oxygen-rich headwaters of streams and to springs.Data logger records over a full year revealed arithmetric means of water tem-peratures for typical habitats of D. balcanicus (e.g., springs below the Troyan pass, Bulgaria) of 6.65°C (range 1.08-15.44°C).This species grazes epilithic algae.

Diagnosis of species of Drusus of the grazer clade lacking areas of spinules or additional spines on head capsule
Mandibles are spoon-shaped (Fig. 1E).The larva of Dru sus balcanicus lacks spinule areas and additional bristles and spines on its head capsule (Fig. 4C; Table 4).Dorsal gills present; basal sclerites of setae on the first abdominal sternum fused to sclerotized plates or form multilobed patterns (Fig. 5B); anterior row of setae present near the dorsal pronotal midline (Fig. 4C); mid and hind legs with dorsal edge setae restricted to distal third of tibiae.It shares these features with Drusus franzressli, D. improvisus, D. nigrescens Meyer-Dür, 1875, D. rectus, D. spelaeus, Ec clisopteryx malickyi Moretti, 1991, Metanoea flavipennis and M. rhaetica.Due to the presence of a low central ridge on the pronotum, the larva of D. balcanicus is similar to that of Drusus franzressli (Table 5), but the latter also has a distinct lateral ridge (Fig. 5F, arrows) which is absent in D. balcanicus (Fig. 4F).In addition, the basal sclerites of the central setae on the first abdominal sternum are fused into a large, uniform central plate (Fig. 5G) in D. franzressli and form a multilobed sclerotized pattern in D. balcanicus (Figs 5B-D).
Head.Head capsule roundish, dark brown with lighter orange areas around foramen occipitale and a smooth surface sculptured by shallow wrinkles (Figs 5H, I).Each parietale with 20-25 long bristles and short, strongly tapering spines plus a standard set of 12 primary setae, mostly anterior and dorsal to the eye.Also on frontoclypeus there are 12-18 long bristles and short, strongly tapering spines, plus standard set of 6 pairs of primary setae, mostly on anterolateral corners (Figs 5H, I).
Thorax.Pronotum dark brown to blackish brown.Pronotal surface relatively smooth, sculptured by shallow wrinkles (Figs 6C, D).Dorsal profile in lateral view with low ridge not elongated laterally.Ridge gently ascending from posterior pronotal border with a distinct step leading down to anterior, lower 2/3 of pronotum (Figs 6B, D).In anterior view with deep central notch flanked by two an-table 4. Synopsis of the characters separating the currently known Drusinae larvae (5th instars) with spoon-shaped mandibles and no spinule areas, additional bristles or spines on the head capsule (i.e., only the standard set of 18 pairs of primary setae is present).2008) Waringer et al. (2008aWaringer et al. ( , 2010Waringer et al. ( , 2011) ) teriorly directed hooks (Figs 6B-D).In total, 60-75 long dark bristles and short, strongly tapering spines are distributed over each pronotal half.Prosternite light brown, with medium brown anterolateral corners, trapezoidal in shape and tapering posteriorly.Mesonotal sclerites dark brown to blackish brown with lateral and posterior margins darkly sclerotized.There are 15-25 setae in anterior setal group sa1, 17-35 in posterior group sa2 and 18-25 in lateral group sa3 (Fig. 6D).
Abdomen.Posterior sclerite present on lateral protuberances.On 1st abdominal sternum, ventral setal areas sa1, sa2 and sa3 fused, creating continuous field of 70-100 setae; basal sclerites of setae in the central area of the first abdominal sternum mostly small and inconspicuous except for four larger basal sclerites near midline and immediately ventral to the lateral protuberances (Fig. 6F).Eighth abdominal dorsum with 2 to 4 long and 4 short posterodorsal setae (pds).Only 1 posterolateral seta is present on each half of 9th abdominal dorsum.Light to medium brown sclerite on 9th abdominal tergum semicircular, with 10 long and several shorter setae along its posterior border, 2 of the long setae in the position of central intermediate c setae.
Habitat.Drusus botosaneanui inhabits springs and the upper regions of the headwaters of streams as well as midstream regions of rivers at 655 to 1450 m above sea level (Ibrahimi et al., 2012).Mean annual water temperatures of the sites inhabited by D. botosaneanui (e.g., tributary of Beli Iskar, Bulgaria) were 5.66°C (annual range 0.08-14.38°C).This species grazes on biofilms and epilithic algae.
General morphology.Larva eruciform, head and sclerotized parts dark brown, nonsclerotised parts whitish.Body length of final instar larva 10.7-12.9mm, head width 1.24-1.34mm.table 5. Synopsis of characters separating the currently known Drusinae larvae (5th instars), which share the following group morphomatrix: Spoon-shaped mandibles; lack of additional head bristles, spines or spinule areas; setae of anterior row present near dorsal pronotal midline; dorsal gills present; setae on dorsal edge restricted to distal third of mid-and hind tibiae; basal sclerites of setae on first abdominal sternum fusing into sclerotized plates or multilobed patterns.Head.Head capsule roundish, dark brown with lighter orange areas around foramen occipitale and with smooth surface sculptured by shallow wrinkles (Figs 7A, B).Setation as in D. botosaneanui.Ventral apotome orange, narrow and parallel-sided; postgenal suture approximately 60-66% of apotome length.

Species
Thorax.Pronotum dark brown to blackish brown.Pronotal surface coarsely granulated with adjacent series of granuli creating ribbed structures (Fig. 7B).Dorsal profile in lateral view with low ridge not elongated laterally.Joint between the posterior and anterior sides of ridge smooth and lacking distinct step; anterior side gently sloping down to anterior part of pronotum (Figs 7B,C,7I).Central notch very shallow, flanking anteriorly directed hooks absent (Figs 7A, C).In total, 60-75 long dark bristles and short, strongly tapering spines scattered over each pronotal half.Prosternite light brown, with medium brown anterolateral corners, trapezoidal in shape and tapering posteriorly.
Habitat.D. tenellus prefers the epi-and metarhithral zone of oxygen-rich streams with high to moderate currents at altitudes >1450 m a.s.l.Mean annual water temperature for sites inhabited by D. tenellus (e.g., Strežimirska reka, Mavrovo, Macedonia) were 6.87°C (annual range 5.11-8.54°C).This species is a grazer of biofilms and epilithic algae.

Diagnosis of species of Drusus of the grazer clade with additional spines on head capsule
Mandibles are spoon-shaped (Fig. 1E).Drusus boto saneanui and D. tenellus belong to the group of Drusinae table 6. Synopsis of characters separating the currently known Drusinae larvae (5th instars) with spoon-shaped mandibles and with additional bristles and spines on the head capsule (in addition to the standard set of 18 pairs of primary setae).

Species
Pronotum with ridge extending laterally to the anterior pronotal margin?

DISCUSSION
Previous studies that associated adults and larvae in caddisflies have used COI (e.g., Waringer et al., 2008b;Graf et al., 2009), COI & WG (Waringer et al., 2013a), COI & 28S rDNAs (Zhou et al., 2007), or COI, WG & LSU sequences (Previšić et al., 2014).The additional use of WG, LSU and CAD did not bring additional information to our 1200 bp long COI sequences.However, the use of unlinked nuclear markers provides independent support for the sorting of mitochondrial lineages, which could also result from historical isolation of presently admixed populations (e.g., Elbrecht et al., 2014).It is thus advisable to use both nuclear and mitochondrial markers for life stage associations.The nuclear genes we used, WG and CAD, proved sufficiently variable to discern species in this study.The level of variation is similar in WG and even higher in CAD compared with COI.Of the two genes we used, CAD performed somewhat better in our study, but both seem suitable for associating life stages of caddisflies.
All four species described in the present paper belong to the largest group of epilithic grazers, which lack terminal teeth on their mandibles (Figs 1D, E, 4D).Based on the presence or absence of bristles and setae in addition to the standard set of 18 pairs of primary setae on the larval head capsule, the grazer clade is separated into three subgroups: Subgroup 1 with an area of spinules posterior to each eye (Figs 1E, F; white ovals).Such spinule areas occur in members of the Drusus bosnicus Group.Marinković-Gospodnetić (1971a) assigned D. bosnicus, D. klapaleki, D. plicatus Radovanović, 1942, D. radovanovici and D. ramae Marinković-Gospodnetić, 1971b to the D. bosnicus Group based on similarity of main structures of the male genitalia.Later, D. krusniki Malicky, 1981, D. medianus, D. septentrionis and D. vespertinus were added to this group (see discussion in Kučinić et al., 2011a).Of these species, the spinules are absent in D. ramae.They are present in the hitherto unknown larva of D. serbicus.
Finally, D. balcanicus belongs to the largest subgroup of the grazer clade, in which only the standard set of primary setae is present on the head capsule (Figs 4C, D).
The spines that define the subgroup 2 (e.g., Figs 5H, I, 7A) have a length of 0.4 mm or more in Ecclisopteryx guttulata and are one magnitude longer than the spinules in Drusus serbicus and associated species measuring up to 0.03 mm.These morphological traits are in line with distinct differences in downstream distribution patterns: species with additional spines on their head capsule (morphological features summarized in Table 6) are most abundant in the epi-and metarhithral section, whereas Drusinae species without spines or with spinules are only found in spring or spring brook sections (eucrenal-hypocrenal).Statzner & Higler (1985) have shown that the eucrenal and hypocrenal sections of streams (source and springbrooks) are frequently characterized by relatively low hydraulic stress.The hypocrenal-epirhithral transition zone is followed by a section with high hydraulic stress, which, after the next zone of transition at the break-point of the slope, is then followed by a zone of lower hydraulic stress (Statzner & Higler, 1985).As Drusinae larvae face into the current (own observation), the presence of spines on the head capsule of this species group summarized in Table 6 may be associated with their presence in such hydrologically high-stress sections within the stream continuum.Videler (1995) has shown that small irregularities in the scales of fish can reduce shear stress in the boundary by a maximum of 10% compared with the shear stress of a smooth surface, a mechanism based on the impedance of cross flow under well-defined conditions.The function of roughness probably reduces total drag by generating premature turbulence and by boundary layer thinning, despite an increased friction over the surface (Videler, 1995).
The adults and larvae of Drusus serbicus, D. botosa neanui and D. balcanicus were sampled in the months of May and June in 2012 and 2013, and of D. tenellus in July 2010.This is in accordance with the reported spring flight period of D. serbicus as the type and paratype specimens were collected on 30 May 1970(Marinković-Gospodnetić, 1971a).Drusus balcanicus is also a spring and early summer species with a rather short flight period, whereas it is longer in D. botosaneanui, which is on the wing from spring to autumn (Graf et al., 2008).A prolonged flight period has also been recorded for D. tenellus, for which adults are still being collected in the first week of October (Oláh & Kovács, 2013).
With respect to distribution, D. balcanicus is a species (micro-) endemic to the eastern Balkan Peninsula where it is restricted to the Stara Planina and Vitosha, whereas D. serbicus is (micro-) endemic to the Dinaric Western Balkans and restricted to the Dinaric Alps.Drusus tenellus has a wider range, with records from the Carpathians and the Dinaric Eastern Balkans.The distribution of D. botosane anui is even wider, covering the Dinaric Western Balkans, the Hellenic and Eastern Balkans as well as Asia Minor.

table 2 .
Characteristics of the molecular data sets used in the phylogenetic analysis and larval-adult associations.
* 11 Ns were added between the two fragments of mtCOI.
1In E. madida, the pronotal ridge is rather sharp and almost concave anteriorly (Figs 7F, G; 8B), in D. schmidi there is a distinct step (Figs 7D, E, H), and in D. tenellus the anterior section of the ridge gently slopes down to the anterior part of the pronotum (Figs 7B, C, I).² In E. keroveci, the number of additional spines on each parietale is 12-20, in E. ivkae 1-7.species