Candona alchichica, 2017

Acosta, Raúl, Prat, Narcís, Ribera, Carles, Michailova, Paraskeva, Hernández-Fonseca, María Del Carmen & Alcocer, Javier, 2017, Chironomus alchichica sp. n. (Diptera: Chironomidae) from Lake Alchichica, Mexico, Zootaxa 4365 (1), pp. 53-70 : 56-63

Chironomus alchichica Acosta & Prat , sp. n.

Type material Holotype: male (preparations in Euparal ), obtained by breeding from larvae collected in Lake Alchichica (50 m deep), state of Puebla, Mexico (19°24.7’ N, 97°24.0’ W), 3.VI.2012 ( CRBA 67541 ), Leg. J. Alcocer . Paratypes: 6 males (CRBA 67542- 67544), 3 pupae (CRBA 67552), 10 larvae ( CRBA 67545- 67551 ) (preparations in Euparal ), same locality as holotype, 02.I.2012, 02.V.2012, 03.VI.2012, 03.VII.2012, 04.XI.2012, Leg. J. Alcocer.

Etymology. This species is named for the type locality of the specimens.

Description. Male imago ( Fig. 1 ) (n= 7). Total length 3.95–5.07, 4.34; wing length 2.99–4.25, 3.53; total length / wing length 1.19–1.41, 1.28; wing length/length of profemur 2.64–3.94, 3.14. Coloration. Head capsule and maxillary palps, yellowish brown. Eyes and antennal flagellum, dark brown. Thorax, dark brown with mesonotal stripes. Abdomen, pale brown, dorsally with dark brown markings on tergites I–VII, wider on tergite VII. Legs with all segments uniformly pale yellow.

Head. Antennal flagellum: 1399–1621.1510; AR=3.75–3.83 3.79. Clypeus with 15–20 (6) setae, 30–40 (5) temporal setae. Palpomere lengths 2–5: 40–55.16, 47.12; 130.16–190, 171.46; 172–205, 188.89; 219.45–255, 244.76.

Wings. Membrane transparent without setae. Alula and axillary area with brown spots. R 2+3 very thin, ends near to edge of wing. Sc, R, R2+3, M1+2, M3+4, Cu, Cu1 and An, pale brown; C, M, R1, R4+5 and RM dark brown. Squama with 20–26 (7) setae. Brachiolum with 2 (9) setae. R, R1 and R4+5 with 20–27 (9); 20–24 (9); 20–24 (8) setae respectively. M without setae. VR =0.91–0.95, 0.94.

Legs ( Fig. 1a ). Segments lengths and main proportions are given in Table 1. Fore tibia with 2–3 thick apical setae. Mid and hind tibial combs with 2–5 short spurs. Pulvilli developed.

Genitalia ( Fig. 1b and c ). Anal point proximally triangular, 61.73–70.88, 65.5. Superior volsella curved in the apex with an expanded knob, 112.68–131.96, 123.36. Gonostylus elongate 178.62–204.85, 195.4 long with 13–15 (7), 6–8 (7) and 6–7 (7) setae in the basal, medium and distal part, respectively. Gonocoxite short, 95.23–118.54, 105.19 long. HR: 0.47–0.64, 0.54. HV: 2.01–2.53, 2.26.

Pupa (n = 3) ( Fig. 2 ).

Mean length of abdomen: 4.9–6.2, 5.4. Cephalothorax: cephalic tubercles conical: 145.2–209.72, 120.52; frontal setae: 46.32–99.6, 70.23; thoracic horn basal ring: 107.42–137.56, 122.44. Thorax is scarcely granulose ( Fig. 2a ). Thoracic chaetotaxy: two precorneal, four dorsocentral and antepronotal setae indistinguishable. Abdomen: without dorsal shagreenation patches evident. Abdominal segment II has a continuous row of hooklets ( Fig. 2b ). The spinules of tergite III–V diminish in size from the posterior to the anterior end. Spinules of tergite II, VI and VIIII are reduced, tergite VII without spinules ( Fig. 2c ). Pedes spurii B and A well developed on segments II and IV respectively. Segments I –IV=0, 3, 3, 3 lateral setae; V–VIII= 4, 4, 4, 5 lateral taeniae. Segment VIII spur with 5–6 apical teeth ( Fig. 2d ). Anal lobe on each side with more than 115 taeniate fringe setae.

Larva (n=10) ( Fig. 3 ).

Body colour (live) bright red, at larva stage IV about 9–10 mm in length, with short posterolateral tubules (LT) on abdominal segment VII ( Fig. 3a ) and two pairs of ventral tubules (VT) on abdominal segment VIII ( Fig. 3b ), the anterior with “elbows” and the posterior coiled. Ventral head length: 0.26–0.31, 0.28. Frontoclypeus light brown or yellowish, with the gular region slightly darker ( Fig. 3c ).

Mandible ( Fig. 3d ). Three very dark teeth (3, 4, 5). The rudimentary 1 st tooth is visible only in certain positions of the mandible. The 2nd and 6th teeth (fused or only partially free) are pale and well seen- a good diagnostic marker of the species. The 6th tooth is fused or only partially free. The pecten mandibularis is very sharp at the apical end.

Antenna ( Fig. 3e ). Consists of 5 segments. Basal antennal segment 105.7–151.67, 118.23 long. The 3rd segment is smaller than the 4th segment, the 5th segment is the smallest. Segments (2–5): 29.69, 8.55, 15.98 and 7.8 long. AR: 1.69–2.39, 1.9. Antennal blade extends to the end of the antenna, 57.76–72.01, 65.8 long. Ring organ (RO) is located on the basal segment about one third of the length of the segment up (0.18–0.29, 0.23). Lauterborn organ (LO) is visible.

Mentum ( Fig. 3f ). Teeth are dark with a median tooth trifid. The accessory tooth (c2) is slightly separated from the median tooth. The 4th lateral tooth is smaller than the 3rd and its height is similar to the 5th. The 6th tooth is pale and is the smallest. The anterior edge of the ventromental plate is smooth ( Fig. 3g ).

Premandible ( Fig. 3h ). Pale, with two teeth. Ventral tooth thinner and slightly longer than the dorsal tooth, which is significantly larger. Pecten epipharyngis consists of a single comb with 13–14 simple teeth.

Diagnostic characters: Adults. The male of C. alchichica sp. n. can now be distinguished from the other previously described species of Chironomus by its relatively small size, antennal ratio (AR) and bristle ratio (BR) of lower than 2, as well as its triangular anal point and its superior volsella (SV) that sharply attenuates to a point with an expanded knob (Type E, according to Strenzke 1959 and Martin 2017).

Pupae. The pupae are smaller than 10mm. The thorax is scarcely granulose and there are no spinules in the pleura of the IV segment. In segments III –V, the spinules of the tergite diminish in size from the posterior to the anterior end. The species is distinguished by the presence of 9–10 spines on the spur of abdominal segment VIII.

Larvae: According to the classification of types from Martin (2017), Proulx et al. (2013), Vallenduk & Moller Pillot (1997) and Webb & Scholl (1985), the larvae of C. alchichica sp. n. is of the “ plumosus - type ” since abdominal segment VIII has long anterior ventral tubules (VT) with “elbows” and a coiled posterior VT. In addition, abdominal segment VII has short lateral tubules. The head capsule is light brown without dark bands in the clypeus and the gula is mostly colourless. The mentum has six pairs of dark lateral teeth, the median tooth is trifid and does not have a basal part more narrow than the median part. The c2 teeth are slightly separated (Type IB), and the 4th lateral teeth are smaller, their height being about equal to that of the 5th lateral teeth (Type II), the last tooth are small. The mandible is Type IA with the 6th tooth (3rd inner) fused and lighter in colour than the others. The ventromental plates have a smooth anterior edge. The antenna has 5 antennomeres, the last one is very small in size, and the 3rd antennomere is shorter than the 2nd and 4th. The Ring organ (RO) is less than one third up from the base of the segment. The AR is higher than 1.5.

Karyological description ( Fig. 4 ). This species belongs to thummi cytocomplex (Keyl 1962), with chromosome arm combinations: AB CD EF G. It has three Balbiani rings (BRs), located on arms B and G, as well as three Nucleolar Organizers (NORs) on arms C, D and G. Those on arms C and D are not always well expressed in all studied cells. The chromosomes are very compact and have high levels of polyteny, which make analysis very difficult. Few cells with good band patterns of the polytene chromosomes were available for analysis. Chromosomes AB and CD are metacentric, chromosome EF is submetacentric, and chromosome G is acrocentric. The centromere regions in chromosomes AB CD and G appeared as dark heterochromatic bands, while those on chromosome EF had the appearance of a thin band ( Fig. 4a ).

Arm A ( Fig. 4b ): Can be distinguished from C. riihimaekiensis via three steps of fixed homozygous inversion.

C. riihimaekiensis : 1 – 2c – 10 – 12 – 3 – 2 d – 9 – 4 – 13 – 14 - 15 – 16 – 17 - 19

Intermediate sequences: 1 – 16 – 15 – 14 – 13 - 4 – 9 – 2 d – 3 – 12 - 10 - 2 c – 17 - 19

Intermediate sequences: 1 – 13 – 14 – 15 – 16 – 4 – 9 – 2 d – 3 – 12 – 10 – 2 c – 17 – 19

C. alchichica sp. n.: 1 – 13 – 14 – 15 – 16 – 2c – 10 – 12 – 3 – 2 d – 9 – 4 – 17 – 19

Arm B ( Fig. 4b ): With BR near to the telomere.

Arm C ( Fig. 4c ): With a constriction near to the telomere (indicated by a small arrow) and a Nucleolar Organizer (NOR) near to the centromere. However, the Nucleolar Organizer (NOR) is not well seen in all studied cells.

Arm D ( Fig. 4c ): With a NOR near to the centromere but not well expressed in all cells.

Arm E ( Fig. 4d ): similar to C. riihimaekiensis but distinguished by fixed homozygous inversion.

C. riihimaekiensis : 1 – 3e – 5 – 10b – 4 – 3f – 10c – 11 – 12- 13

C. alchichica sp. n.: 1 – 3e - 10c – 3f – 4 – 10b – 5 – 11 – 12 – 13

Arm F ( Fig. 4d ): similar to the band patterns of C. riihimaekiensis

C. alchichica sp. n.: 1 – 8c – 12 – 17 – 10 – 8 d – 11 – 18 – 23

Arm G ( Fig. 4e ): Has one Nucleolar Organizer (NOR) located in one telomere region, and two Balbiani Rings (BRs). The chromosome is partly unconjugated.

There is no inherited inversion polymorphism. In material from a depth of 30m, a somatic inversion on arm F was observed in a single individual.

The molecular identity of C. alchichica sp. n. ( Fig. 5 , Table 2). Specimens, localities and sequences analyzed in the present study are listed in Appendix 1 with their corresponding GenBank accession numbers. The matrix used in the phylogenetic analysis includes 58 terminals and 696 aligned characters. Figure 5 shows the maximum likelihood tree obtained using partial sequences of the cox1 gene. Chironomus alchichica sp. n. clusters together with its sister species C. decorus (bootstrap support = 92%). Both species are the sister group of C. bifurcatus (93%), and this evolutionary line is the sister group of C. maturus .

BETwEEN SPECIES WIThIN SPECIES C. maturus C. bifurcatus C. decorus

C. maturus 0.001 C. bifurcatus 0.116 0.016 C. decorus 0.09 0.069 0.004 C. alchichica sp. n. 0.086 0.067 0.03 0.004 The evolutionary divergence over sequence pairs (uncorrected p-distances) of cox1 between C. alchichica sp. n. and its sister species C. decorus is 3% ( Table 2). The uncorrected p-distances between the new species and C. bifurcatus and C. maturus are 6.7% and 8.6% respectively. The average internal distances within each species are low ( C. alchichica sp. n. 0.4%; C. decorus 0.4; C. bifurcatus 1.6 and C. maturus 0.1).

Habitat and ecological notes. C. alchichica sp. n. inhabits depths from the shallow littoral area down to the deepest portion of the lake (62 m), suggesting a tolerance of a broad range of environmental variables (Table 3). The littoral area where the species was found is sandy and rich in organic matter, and ranged from sediments without vegetation to some that were completely covered with macrophytes (i.e., Zoostera marina and Cyperus laevigatus ) and/or benthic algae (filamentous chlorophytes and cyanobacteria, diatoms) (Ramírez-García & Novelo 1984). The deep benthic zone has fine sediments dominated by clayey silts with high organic matter content (algae detritus), while higher aquatic plants are absent in the aphotic depths. The water is clear, with temperatures varying from comparatively warm (18–25°C) in the littoral, to cold (down to 14°C) in the deepest part of the lake. Dissolved oxygen content was also variable ranging from 6.5–9.1 mg L– 1 in the littoral, and from 0.9–5.8 mg L– 1 in the hypolimnion (Alcocer & Bernal-Brooks 2010).

TABLE 3. Averages (± standard deviation) and ranges of environmental variables of the littoral and profundal zone of Lake Alchichica, Puebla, where C. alchichica sp. n. is present (T = temperature, K25 = electric conductivity, DO = dissolved oxygen, OM = sediment organic matter, CO3 = sediment carbonates, Text = sediment texture).

ZONE T K25 DO PH OM CO3 TExT

(°C) (MS CM -1) (MgL -1) (%) (%) (ϕ)

LITTORAL 20.7±1.0 12.6±0.6 8.3±1.6 9.0±0.0 5.6±3.6 11.2±1.7 1.4±0.0 18.3–24.9 11.0–13.2 6.5–12.3 8.9–9.0 2.8–8.4 1.9–29 0.2–2.3

PROFUNDAL 14.4±0.0 13.6±0.7 2.5±2.0 9.2±0.1 34.7±3.5 13.4±4.5 5.9±1.2 14.4–14.5 11.9–14.5 0.9–5.8 9.0–9.6 28.9–40.1 6.2–24.5 3.0–9.0 It should be noticed that while C. alchichica sp. n. is present all year around in the littoral zone, it was only present in the depth zone from January (or February) to April (or May), during the circulation to early stratification periods respectively, and during the same period when the bottom water remained oxygenated, as explained by Hernández et al. (2014). In the profundal zone, C. alchichica sp. n. shares its habitat with just one other species, the ostracod Candona patzcuaro (Hernández et al. 2014) , while in the littoral zone up to 20 different taxa are present, although the oligochaete Limnodrillus hoffmeisteri and the amphipod Hyalella azteca widely dominate (Alcocer et al. 2016).

Chironomus alchichica sp. n. could become the 9th endemic species described for Lake Alchichica, which include the diaptomid copepod Leptodiaptomus garciai (Montiel-Martínez et al. 2008; Osorio-Tafall 1942), the atherinid fish Poblana alchichica (De Buen 1945) , the hemipteran Krizousacorixa tolteca (Jansson 1979) , the salamander Ambystoma taylorii (Brandon et al. 1981) , the isopod Caecidotea williamsi (Escobar-Briones & Alcocer 2002) , the diatom Cyclotella alchichicana (Oliva et al. 20 06), the harpacticoid copepod Cletocamptus gomezi (Suárez-Morales et al.. 2013), and the candonid ostracod Candona alchichica (Cohuo et al. 20 17).

Figures

FIGURE 1. Chironomus alchichica sp. n. ADULT:A) FORE TIBIA AND FIRST TARSAL SEgMENT (ThE ARROw INDICATES ThE APICAL SETAE);B) SUPERIOR VOLSELLA wITh ThE APEx ExPANDED;C) HyPOPygIUM.BAR = 50 µM.

FIGURE 2. Chironomus alchichica sp. n. PUPA: A) ThORAx wITh gRANULOSE AREA REDUCED; B) ABDOMINAL SEgMENT II wITh A CONTINUOUS ROw OF hOOKLETS; C) ABDOMINAL SEgMENT III wITh AREA OF SPINULES DIMINIShINg IN SIzE FROM ThE POSTERIOR TO ANTERIOR EDgE;D) SPUR OF ABDOMINAL SEgMENT VIII. BAR = 100 µM.

FIGURE 3. Chironomus alchichica sp. n. LARVA:A) POSTEROLATERAL TUBULES (LT) ON ABDOMINAL SEgMENT VII; B) VENTRAL TUBULES (VT) ON ABDOMINAL SEgMENT VIII; C) HEAD CAPSULE; D) MANDIBLE; E) ANTENNA: RINg ORgAN (RO), LAUTERBORN ORgAN (LO); F) MENTUM g) VENTROMENTAL PLATE;h) PREMANDIBLE AND PECTEN hyPOPhARyNgIS.BAR = 100 µM.

FIGURE 4. SALIVARy gLAND ChROMOSOMES OF Chironomus alchichica sp. n. A) ChROMOSOMES AB CD EF G ARE VERy COMPACT, wITh DARK CENTROMERE REgIONS OF ChROMOSOMES AB CD G, CENTROMERE REgION OF EF AS A ThIN BAND.NUCLEOLAR ORgANIzER (NOR); BALBIANI RINg (BR). LARgE ARROw INDICATES ThE CENTROMERE REgIONS; B) ChROMOSOME AB; C) ChROMOSOME CD wITh CONSTRICTION IN ARM C (SMALL ARROw) AND TwO NORS IN ARMS C AND D; D) ChROMOSOME EF; E) ChROMOSOME OF ARM G wITh A NOR AND TwO BRS.BAR = 100 µM.

FIGURE5. MAxIMUM LIKELIhOOD TREE OF ThE Chironomus alchichica sp. n., C.decorus (INCLUDINg C. decorus-group SP.2, C. CF. decorus AND C. CF. decorus 2g), C. bifurcatus AND C. maturus, USINg PARTIAL cox1 gENE SEqUENCES. NUMBERS NExT TO NODES CORRESPOND TO BOOTSTRAP SUPPORT VALUES. NUMBERS OF ThE TERMINALS CORRESPOND TO ThE GENBANK ACCESSION NUMBERS. Goeldichironomus devineyae wAS USED TO ROOT ThE TREE.