Diptera: Chironomidae: Orthocladiinae)

Studi Trent. Sci. Nat., Acta Biol., 82 (2005): 69-76 ISSN 0392-0542 © Museo Tridentino di Scienze Naturali, Trento 2006

Thoracic horn structure in Orthocladiini pupae (Diptera: Chironomidae: Orthocladiinae)

Laura Marziali1* & Bruno Rossaro2

1Sezione di Zoologia degli Invertebrati e Idrobiologia, Museo Tridentino di Scienze Naturali, Via Calepina 14, I-38100 Trento, Italy 2Dipartimento di Biologia, Sezione di Ecologia, Università degli Studi di Milano, Via Celoria 26, I-20133 Milano, Italy *Corresponding author e-mail: [email protected]

SUMMARY - Thoracic horn structure in Orthocladiini pupae (Diptera: Chironomidae: Orthocladiinae) - The pupal thoracic horn in Chironomidae is a respiratory organ specialized for oxygen adsorption and the horn surface was suggested as a measure of a species’ oxygen requirements. This structure was analyzed in 31 species belonging to the tribe Orthocladiini within the genera Cricotopus, Eukiefferiella, Orthocladius and Tvetenia. Optima for dissolved oxygen and water temperature were calculated for each taxon using information from a large database. The species with extensive horn are not necessarily the most resistant to low oxygen levels and high temperature and no significant correlation was detected between horn surface and species optima. The species of the genus Cricotopus, characterized by reduced thoracic horn, tolerate lower oxygen levels than Orthocladius: it is suggested that higher tolerance is bound to more frequent respiratory undulations. At taxonomic levels below tribes behavioural and physiological adaptations more than morphological ones may explain the results.

RIASSUNTO - Il corno toracico pupale nelle pupe degli Orthocladiini (Diptera: Chironomidae: Orthocladiinae) - Il corno toracico pupale dei chironomidi è un organo respiratorio specializzato nell’assorbimento di ossigeno e in passato la sua superficie è stata interpretata come misura del fabbisogno di ossigeno di una specie. Questa struttura è stata analizzata in 31 specie all’interno della tribù degli Orthocladiini appartenenti ai generi Cricotopus, Eukiefferiella, Orthocladius e Tvetenia e confrontata con i valori ottimali di ossigeno disciolto e temperatura calcolati per ogni taxon a partire da un’estesa banca dati. Si è potuto constatare che i taxa più resistenti a bassi livelli di ossigenazione e a temperature elevate non presentano necessariamente corni più estesi e non è emersa una relazione statistica significativa tra la superficie del corno e le variabili ambientali misurate. L’elevata resistenza all’ipossia delle specie del genere Cricotopus, caratterizzate da corni ridotti rispetto alle specie del genere Orthocladius, viene attribuita alla maggiore frequenza dei movimenti respiratori delle pupe al variare della temperatura. A livello tassonomico inferiore alla tribù adattamenti comportamentali e fisiologici più che morfologici potrebbero spiegare i risultati ottenuti.

Key words: pupal stage, hypoxia, species tolerance, respiratory movements, Italian waters Parole chiave: stadio pupale, ipossia, tolleranza delle specie, movimenti respiratori, corpi idrici italiani

1. INTRODUCTION dominantly isolated within a silken tube produced by the final instar larva. Moreover, the pupal morphology The pupal stage of chironomids has been long dis- is limited by the shape of the larva in which it devel- cussed in terms of functional and phylogenetic signif- ops and of the adult which develops within it (Lang- icance. Although very short, rarely exceeding 72 hours ton 1989). in duration, the pupal stage is rather critical in chirono- Nevertheless, chironomid pupae are characterized mid life cycle, involving the development of the adult by a great morphological diversity, which allow the (Langton 1995). identification to species, even where larvae or adults The pupa neither feeds nor reproduces and for most are inseparable, as Cricotopus (Hirvenoja 1973). Thus, species its movements are limited to respiratory undu- the pupal stage must undergo evolutionary change inde- lation and ecdysis. In fact, except for the free-living pendently of the other life stages, as discussed by Coff- Tanypodinae and Podonominae, most pupae are pre- man (1979), and it must be highly functional, adapt- 70 Marziali & Rossaro Thoracic horn in Orthocladiini (Diptera: Chironomidae)

Fig. 1 - Pupal thoracic horn of Conchapelopia pallidula (Tanypodinae) (A), Orthocladius excavatus (Orthocladiinae) (B), Tanytarsus sylvaticus (Tanytarsini) (C), Polypedilum nubeculosum (Chironomini connectentes) (D), Paracladopelma camp- tolabis (E) and Chironomus anthracinus (F) (Chironomini genuinae). Fig. 1 - Corno toracico pupale di Conchapelopia pallidula (Tanypodinae) (A), Orthocladius excavatus (Orthocladiinae) (B), Tanytarsus sylvaticus (Tanytarsini) (C), Polypedilum nubeculosum (Chironomini connectentes) (D), Paracladopelma camp- tolabis (E) e Chironomus anthracinus (F) (Chironomini genuinae). ing rapidly in absence of traditional selection forces indeed that along the longitudinal gradient in rivers (Langton 1989). cold stenothermic Diamesinae and Orthocladiinae are Among the structures associated with the mainte- predominant in the upper reaches, while Chironom- nance of the developing adult, the thoracic horn is an inae tolerating higher temperatures and lower oxy- extension of the surface area specialized for oxygen ad- gen concentrations prevail in potamal communities sorption, in addition to body cuticle (Cranston 1995). (Pinder 1995). The littoral zone of lakes is dominated The higher phylogeny of the Chironomidae involves by a fauna similar to that of the rhithral zone of riv- changes in this structure, as the loss of a direct con- ers, and in particular by Orthocladiinae; the profun- nection to the adult tracheal spiracle (Buchonomyiinae, dal, where oxygen may be lacking, is colonized main- Diamesinae, Orthocladiini, Tanytarsini) and the rees- ly by Chironominae. Tanytarsini with simple filamen- tablishment of a secondary indirect connection (Pro- tous horns can prevail in oligotrophic well-oxygenat- diamesinae, Chironomini, Pseudochironomini) (Coff- ed lakes, while Chironomini with plumose organs are man 1979, Rossaro 1988). In free-living species the found in eutrophic conditions (Brundin 1974, Wied- thoracic horn is tubular with a distinctive apical respi- erholm 1980). Therefore, the morphology of the tho- ratory surface, the plastron plate (Tanypodinae); when racic horn could be directly related to the physical and lost, the remnant inner chamber is termed atrium (Po- chemical characteristics�� of the habitat, at least con- donominae). This is lost in Diamesinae and Orthocla- sidering tribes. Currently few information are avail- diinae, which often lack the complete organ. In con- able at genus and species level, but previous studies trast, pupae of Chironominae are characterized by mul- emphasized that also behavioral and physiological ad- tifilamentous and plumose thoracic horns, which act as aptations of species should be considered (Int�� Panis gills (Langton 1995) (Fig.1). et al. 1996; Usseglio-Polatera et al. 2000; Marzia- In general, pupae with reduced or absent thoracic li et al. 2006)��. As regard the respiratory movements horn inhabit oxygen-rich habitats, while species tol- of Chironominae larvae, undulation frequency was erant to hypoxia have extensive respiratory organs shown to increase with decreasing oxygen concentra- (Coffman 1979; Langton 1995). Thus, the horn sur- tion�� (Walshe 1947; Heinis & Crommentuijn 1989). A face was suggested as a measure of a species’ oxy- large body size can contribute to the effectiveness of gen requirements (Thienemann 1954). It was observed driving water through the tube (Int Panis et al. 1996). Studi Trent. Sci. Nat., Acta Biol., 82 (2005): 69-76 71

On the other hand, studies on the physiological devic- 2. METHODS es used by species to survive adverse environmental conditions are still scarce (Rosenberg 1992). Many field surveys were realized by the senior au- These aspects were investigated in Chironomini thor in different freshwater habitats in Italy from the (Marziali et al. 2006), but in Orthocladiini they are Sixties. Chironomid larvae, pupae and pupal exuviae less evident and therefore often still unknown. The�� were collected using Surber and Brundin nets. Differ- aim of this study is to analyze the thoracic horn struc- ent physical and chemical parameters were measured: ture in Orthocladiini pupae in relation with each spe- water temperature, dissolved oxygen, conductivity, pH, cies’ physico-chemical tolerance. Some preliminary nutrients. Samples were slide mounted and identified at observations on respiratory behavior of species are species group or species level, according to the follow- presented. ing keys: Ferrarese & Rossaro (1981), Rossaro (1982),

Tab. 1 - Number of measured specimens (N), mean and standard deviation (st. dev.) of abdomen length (AbdL) and thoracic horn area (ThA), ThA/AbdL ratio, weighted mean of water temperature (Temp) and dissolved oxygen (O2) for each species analyzed. Tab. 1 - Numero di individui misurati (N), media e deviazione standard (st. dev.) della lunghezza dell’addome (AbdL) e del- l’area del corno (ThA), rapporto ThA/AbdL, media ponderata della temperatura dell’acqua (Temp) e dell’ossigeno disciolto

(O2 ) per ogni specie analizzata. Species AbdL AbdL ThA ThA ThA/ Temp O Species Author N 2 Genus group mean μ��m� st. dev. mean μ��m�2 st. dev. AbdL μm °C mg l-1 Cricotopus bicinctus bicinctus (Meigen 1818) 31 2390.3 319.4 944.3 389.9 0.4 18.1 8.7 Cricotopus bicinctus vierriensis Goetghebuer 1935 8 1990.0 522.2 2308.0 673.0 1.2 19.5 9.2 Cricotopus fuscus fuscus (Kieffer 1909) 6 2765.0 259.8 951.1 400.2 0.3 11.8 10 Cricotopus Isocladius sylvestris (Fabricius 1794) 17 3214.5 347.6 2460.6 798.6 0.8 11.1 10.5 Cricotopus tremulus annulator Goetghebuer 1927 16 2445.8 332.4 862.1 345.7 0.4 14.9 10.3 Cricotopus tremulus curtus Hirvenoja 1973 5 2303.5 292.3 1731.7 736.9 0.8 7.5 13.5 Cricotopus tremulus tremulus (Linnaeus 1758) 19 2544.3 242.9 356.2 158.1 0.1 11.4 9.2 Cricotopus tremulus triannulatus (Macquart 1826) 15 2301.0 248.5 984.4 412.8 0.4 13.9 8.6 Cricotopus trifascia trifascia Edwards 1929 14 2709.5 266.2 764.1 408.7 0.3 13.5 10.1 Cricotopus tibialis (Meigen 1804) 6 3064.0 596.7 1387.6 277.9 0.5 7.4 11 Eukiefferiella Akiefferiella ilkleyensis (Edwards 1929) 13 1716.6 226.8 2285.1 501.9 1.3 14.1 8.6 Eukiefferiella claripennis brevicalcar (Kieffer 1911) 13 1584.0 226.3 1856.2 372.5 1.2 4.4 9.2 Eukiefferiella claripennis claripennis Lundbeck 1898 16 1692.9 149.0 2008.6 317.2 1.2 14.8 9.9 Eukiefferiella claripennis fuldensis Lehmann 1972 9 1347.0 162.5 502.2 140.3 0.4 4.7 9.3 Eukiefferiella claripennis lobifera Goetghebuer 1934 13 1651.3 216.8 1411.9 733.7 0.9 7.5 11 Eukiefferiella claripennis tirolensis Goetghebuer 1938 6 1622.0 190.7 1700.3 795.5 1.0 5.2 10.2 Eukiefferiella cyanea Thienemann 1936 7 1838.8 177.2 2020.8 457.1 1.1 4.2 9.1 minor- Eukiefferiella longicalcar minor (Edwards 1929) 6 2139.0 283.3 4239.1 2776.6 2.0 6.2 9.9 Eukiefferiella clypeata (Kieffer 1923) 9 1904.5 290.6 1732.4 417.4 0.9 11.2 10.3 Orthocladius excavatus excavatus (Kieffer 1923) 49 3283.3 354.9 4879.3 1373.0 1.5 16.8 9.1 Orthocladius frigidus frigidus (Zetterstedt 1838) 17 3198.7 407.1 5226.5 2433.0 1.6 5.7 9.2 Orthocladius oblidens oblidens (Walker 1856) 45 2943.3 389.0 4530.4 1440.0 1.5 8 10.6 Orthocladius pedestris pedestris Kieffer 1911 39 3005.0 306.0 8296.4 1655.3 2.8 17 8.4 Orthocladius rhyacobius rhyacobius Kieffer 1911 53 3096.9 359.3 5841.7 1670.4 1.9 11.5 10.2 Orthocladius rubicundus rubicundus (Meigen 1818) 81 2594.2 320.2 2543.3 872.4 1.0 12.7 9.7 Orthocladius ruffoi ruffoi Rossaro & Prato 1992 11 3000.6 386.5 2959.1 1263.5 1.0 14.7 9.2 Orthocladius wetterensis wetterensis Brundin 1956 16 3074.9 265.5 4175.8 1200.4 1.4 9.9 9.8 Tvetenia calvescens (Edwards 1929) 40 1960.3 276.2 2151.2 687.6 1.1 10.4 10 Tvetenia discoloripes Goetghebuer 1936 7 2970.6 278.9 6194.4 986.7 2.1 12.2 9.2 Tvetenia bavarica (Goetghebuer 1934) 22 1723.0 208.6 1188.0 281.0 0.7 6 8.7 Tvetenia verralli (Edwards 1929) 17 2103.5 250.0 2942.9 813.2 1.4 12.1 10.3 72 Marziali & Rossaro Thoracic horn in Orthocladiini (Diptera: Chironomidae)

Ferrarese (1983), Wiederholm (1983, 1986), Langton ghted means can be interpreted as optimum values of & Visser (2003). Slides are deposited in the chirono- environmental variables for each taxon. mid collection of the Department of Biology, Univer- Behavioral tests were realized using pupae of Or- sity of Milan. Data were filed in a relational Microsoft thocladius excavatus Brundin, 1947 and Cricotopus Access® database and are available at web site http:// bicinctus (Meigen, 1818) collected monthly in 2002 users.unimi.it/~roma1999/rossaro.html. in the Ticino River (North Italy, Castelletto di Cuggio- The most abundant and frequent species belong- no, NO) using Surber nets. Samples were transport- ing to the genera Tvetenia, Eukiefferiella, Orthocladi- ed alive to the laboratory and reared in Petri dishes in us and Cricotopus were considered (Tab. 1). Thoracic a thermostatic chamber at 12 °C in controlled condi- horn area and abdomen length of each prepared pupal tions. Each dish was removed at a time from the cham- exuvia were measured using ��a Leica�� DM LB2 micro- ber and temperature was monitored while increasing up scope by means of a Leica DC 180 camera and Leica�� to 25 °C. Pupal undulations were counted while tem- IM 1000® package. perature was rising and the number of movements per Female and male pupal exuviae of each species col- minute was determined. lected in different sites and dates were considered for intraspecific variation. The mean value of horn area and abdomen length 3. RESULTS was calculated for each species and it was correlated with the weighted means of environmental variables 3.1. Morphological analysis of species (water temperature and dissolved oxygen)��, calculated according to the following formula: In all, 31 species were considered, 10 belonging to the genus Cricotopus, 9 to Eukiefferiella, 8 to Or- n thocladius and 4 to Tvetenia (Tab. 1). Depending on ∑ yjkzij − ______i=1 the number of slides present in the collection, from 5 (1) zjk= n ∑ y to 53 individuals per species were analyzed. i=1 ik In general, pupae of Cricotopus and Eukiefferiella where zij is the value of the environmental variable j had less extensive horns than Orthocladius and Tvete- measured in a site i, yik is the abundance of the species nia (Fig. 2). Abdomen length was similar in all species − k in the same site i and zjk is the weighted mean value except for Eukiefferiella, Tvetenia calvescens (Edwards, calculated for species j and the environmental varia- 1929) and Tvetenia bavarica (Goetghebuer, 1934), ble k. According to Ter Braak & Prentice (1988), wei- which were significantly smaller than the others.

Fig. 2 - Mean thoracic horn area (�μ��m2, grey squares) and standard deviation (�μ��m2, black lines) for each considered species of Cricotopus (= C.), Eukiefferiella (= E.), Orthocladius (= O.) and Tvetenia (= T.). Fig. 2 - Media (μm2, quadrati grigi) e deviazione standard (μm2, linee nere) dell’area del corno toracico delle specie analizzate appartenenti ai generi Cricotopus (= C.), Eukiefferiella (= E.), Orthocladius (= O.) e Tvetenia (=T.). Studi Trent. Sci. Nat., Acta Biol., 82 (2005): 69-76 73

Tab. 2 - Intraspecific variation of pupal abdomen length and thoracic horn area for five species of Orthocladius. Tab. 2 - Variazione intraspecifica della lunghezza dell’addome e dell’area del corno toracico pupale per cinque specie di Orthocladius.

Sampling Abdomen length Thoracic horn Species River Sampling site N. specimens date μ�m area μ��m�2 Castelletto di O. excavatus Ticino 22/02/2001 9 2527.1-3577.3 3284.0-7634.2 Cuggiono, NO O. excavatus Adda Rivolta d’Adda, CR 04/12/2001 12 3044.6-3854.0 3716.4-6129.3 O. pedestris Taro Bedonia, PR 15/05/2001 11 2603.8-3359.8 6975.5-9955.6 O. pedestris Taro Compiano, PR 23/04/2002 10 2392.8-3133.2 3934.4-9196.9 Acquanegra sul O. rhyacobius Chiese 14/04/1994 9 2378.8-2951.6 3010.6-6017.9 Chiese, BS O. rhyacobius Taro Compiano, PR 10/01/2002 8 2799.1-3758.5 3748.4-9748.7 O. rubicundus Adda Rivolta d’Adda, CR 12/04/2001 9 2496.5-3031.1 2286.7-3650.6 O. wetterensis Taro Compiano, PR 10/01/2002 7 2674.5-3355.2 2578.5-5666.1

Standard deviation was low for abdomen length a high ThA/AbdL. These species did not emphasize (149.0 - 596.7 μm) and high for thoracic horn surface a positive trend. (140.3 - 2776.6 μm2). In particular, a high intraspecif- Eukiefferiella and Orthocladius species tolera- ic variation was detected for the horn area in Ortho- ting less oxygenated waters had a higher ThA/AbdL cladius species and Eukiefferiella minor ��(Edwards, (Fig. 3). E. minor, Orthocladius rhyacobius Kieffer, 1929) (Fig. 2). 1911 and O. frigidus were exceptions having a high The analysis of specimens of the same species col- ThA/AbdL and being intolerant to low oxygen levels; lected in the same site and date emphasized a high the oxygen tolerant Eukiefferiella fuldensis Lehmann, intraspecific variation (Tab. 2): pupal exuviae of 1972, O. rubicundus and O. ruffoi had a low ThA/Ab- some species exhibited a great variability for abdo- dL. Cricotopus and Tvetenia species showed an oppo- men length and horn area (e.g., Orthocladius exca- site trend, with a high ThA/AbdL in waters with high vatus, O. pedestris Kieffer, 1911, O. rhyacobius Ki- oxygen levels. effer, 1911) . No correlation between ThA/AbdL and environ- A significant correlation between abdomen length mental variables was emphasized considering neither and horn surface was observed (r = 0.7, p <0.01, N = species groups nor genera. 626). Therefore, the ratio between horn area and ab- Cricotopus could tolerate water temperature up to domen length (ThA/AbdL) was considered to remove 28 °C (C. bicinctus, North Italy, Po River [Isola Se- the influence of body size on horn surface. rafini, CR], 05/07/1982) and dissolved oxygen down to 2.2 mg l-1 (C. annulator, North Italy, Lambro Ri- 3.2. Relationship between species morphology and ver [Lambrugo, MI], 10/06/1996) and had a ThA/Ab- environmental variables dL = 0.47 µm, with a mean abdomen length of 2531.7 µm and a mean horn area of 1200.8 µm2. Orthocla- The mean value of ThA/AbdL for each species dius was never found over 26.5 °C (Orthocladius obli- within a genus was plotted against the weighted means dens (Walker, 1856), North Italy, Ticino River [Turbi- of water temperature and dissolved oxygen (Fig. 3). go, NO], 26/06/2003) and under 6 mg l-1 of dissolved There was a general positive trend in increasing ThA/ oxygen (O. oblidens, North Italy, Adda River [Lanca AbdL with water temperature, but no significant cor- delle Due Acque, LO], 25/06/2002) and had ThA/Ab- relation was detected. C. bicinctus, Cricotopus an- dL = 1.66 µm, with a mean abdomen length of 3071.7 nulator Goetghebuer, 1927, Cricotopus trifascia Ed- µm and a mean horn area of 5088.8 µm2. wards, 1929, Cricotopus tremulus (Linnaeus, 1758), Thoracic horns of Eukiefferiella, Orthocladius and Orthocladius ruffoi Rossaro & Prato, 1992, O. rubi- Tvetenia species colonizing lotic habitats were more cundus, O. excavatus tolerating high water tempera- extensive than the respiratory organs of Cricotopus spe- ture had a low ThA/AbdL, whereas Cricotopus cur- cies living in waters with slow current (e.g., C. bicinctus Hirvenoja, 1973, E. minor and Orthocladius fri- tus, C. fuscus (Kieffer, 1909), C. sylvestris (Fabricius, gidus (Zetterstedt, 1838) inhabiting cold waters had 1794), C. tremulus) (Thienemann 1954). 74 Marziali & Rossaro Thoracic horn in Orthocladiini (Diptera: Chironomidae)

Fig. 3 - Relationship between weighted mean of water temperature (left column) or dissolved oxygen (right column) and thoracic horn area / abdomen length ratio (= ThA/AbdL) for species belonging to the genera Cricotopus, Eukiefferiella, Or- thocladius and Tvetenia. Black lines = minimum square lines. Fig. 3 - Relazione tra la media ponderata di temperatura dell’acqua (colonna sinistra) o di ossigeno disciolto (colonna destra) e il rapporto tra area del corno e lunghezza dell’addome (= ThA/AbdL) per alcune specie appartenenti ai generi Cricotopus, Eukiefferiella, Orthocladius e Tvetenia. Linee nere = rette dei minimi quadrati. Studi Trent. Sci. Nat., Acta Biol., 82 (2005): 69-76 75

3.3. Behavioural tests Within the genus Cricotopus very tolerant species with reduced thoracic horn and size were observed, The number of respiratory movements performed such as C. bicinctus, C. annulator, C. trifascia, C. tre- by pupae of C. bicinctus and O. excavatus was compa- mulus (Hirvenoja, 1973). Within the genus Orthocla- red at different water temperature (Fig. 4). dius, O. ruffoi, O. rubicundus and O. excavatus, living In all tests the number of undulations per minute in the potamal zone of rivers, were the most tolerant was shown to increase rapidly with water temperatu- (Rossaro & Casalegno 2001). The cold stenothermal re, from 0 at 12 °C to more than 150 at 22 °C. In sin- C. curtus, E. minor and O. frigidus had extensive tho- gle specimens this number was constant in time at the racic horn (Rossaro 1979). same temperature. At temperature higher than 22 °C Cricotopus species with low ThA/AbdL were tole- respiratory movements became less frequent till dea- rant to warm waters with low dissolved oxygen. Beha- th after 24-48 hours. vioural or physiological devices were supposed to be Pupae of C. bicinctus performed more frequent un- adopted by these species to survive, in contrast with Or- dulations (about 50 movements more) than pupae of thocladius species. In fact, pupae of C. bicinctus per- O. excavatus (Fig. 4). formed high frequency undulatory movements, whereas pupae of O. excavatus did not. Physiological adaptations in Orthocladiini could play an important role in determine the tolerance ran- ge of each species; at present few information are available (Rosenberg 1992; Berra et al. 2004).

5. CONCLUSIONS

The thoracic horn of chironomid pupae was analyzed as an extension of the respiratory surface in relation to oxygen levels in lacustrine Chironomini spe- Fig. 4 - Mean number of respiratory movements performed cies. A significant correlation between the extension per minute by pupae of Cricotopus bicinctus and Orthocla- of the horn and the tolerance limits of species was de- dius excavatus at different water temperature. tected in previous researches (Marziali et al. 2006). In Fig. 4 - Numero medio di movimenti respiratori effettuati al minuto dalle pupe di Cricotopus bicinctus e Orthocladius the present work no clear relationship between the horn excavatus a diverse temperature dell’acqua. surface and environmental variables was emphasized within the tribe Orthocladiini. It is possible that point measurements of environmental variables are not pro- 4. DISCUSSION per to define the highly variable environmental conditions in which these animals live. Another explanation The surface of thoracic horn was shown to be hi- may be that at taxonomic levels below tribes behaviou- ghly variable within and between species. The positi- ral and physiological adaptations are more important ve correlation between abdomen length and horn sur- than morphological ones. face emphasized that this organ may act as an extension of respiratory surface to balance the surface/vo- lume ratio of animals. ACKNOWLEDGMENTS No significant correlation was detected between the ratio of thoracic horn surface to abdomen length (ThA/ This research was supported by a grant from Ita- AbdL) of species and environmental variables (wa- lian MURST within the project “An analysis of spa- ter temperature and dissolved oxygen). This contra- tio-temporal distribution of species and populations dicts the well known statement that species with more belonging to critical groups living in inland waters, extensive horn area colonize warmer waters with less with morphological and molecular characterization” dissolved oxygen. In fact, it is well known that Chiro- n. 2002058154_002. The authors wish to thank Valeria nomini genuinae have a multibranched horn and tole- Lencioni, Curator of the Section of Invertebrate Zoo- rate lower oxygen level than Chironomini connecten- logy and Hydrobiology, Museo Tridentino di Scienze tes, Tanytarsini, Orthocladiini and Diamesini (Marzia- Naturali, Trento (Italy), and an anonymous referee for li et al. 2006). the helpful suggestions. 76 Marziali & Rossaro Thoracic horn in Orthocladiini (Diptera: Chironomidae)

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