Botryocladius gen. n.: a new transantarctic genus of orthocladiine midge (Diptera: Chironomidae)

Peter S. Cranston1 and Donald H.D. Edward2 1CSIRO Entomology, G.P.O. Box 1700, Canberra, A.C.T. 2601, Australia. 2Department of Zoology, The University of Western Australia, Nedlands, Western Australia 6907

Abstract. A new genus of orthocladiine Chironomidae, Botryocladius (type species B. grapeth sp. n. from eastern Australia) is described and illustrated in all life history stages. All thirteen included species are described as new, six from eastern Australia (B. grapeth, B. brindabella, B. mdfrc, B. collessi, B. tasmania, B. australoalpinus), two from Western Australia (B. bibulmun and B. freemani), one from ephemeral streams on rock outcrops in western, central and eastern Australia (B. petrophilus) and four from Patagonian Argentina and Chile (B. edwardsi, B. glacialis, B. mapuche and B. tronador). All Australian species are known from at least pupal exuviae, most from adult males, and several from larvae. In contrast, only B. edwardsi amongst Neotropical species is known from the adult male: all others are described from pupal exuviae. The immature stages are lotic in Australian permanent and temporary streams and Patagonian glacial streams and rivers, and lentic in Neotropical glacial-fed and Australian subalpine lakes. Botryocladius appears to belong with a grouping centred on two formally undescribed taxa from Australia. The genus evidently demonstrates a vicariant distribution with at least two sister-group relationships between South American and Australian taxa, providing a minimum dating for the clade of 38 m.y.b.p., with apparent absence from New Zealand indicating a maximum date of 80 m.y.b.p..

Introduction

The evidence for extant insect genera being shared between Australia have been examined and eliminated from widely separated Gondwanan land masses is accumulating, not consideration. least for the non-biting midges (Chironomidae) (e.g. Freeman, Finally we discuss the biology, biogeography and phylogeny. 1961; Brundin, 1966). However, the increasingly well- For the subfamily Orthocladiinae the phylogeny is so uncertain understood vicariant patterns shown by the chironomid faunas that Sæther (1990a) suggested that orthocladiine genera had of the southern continents have been recognised predominantly character distributions “in such a mess” as to render it from systematic studies concerning only three of the subfamilies impossible to find any pattern of relationships. In that study, a of Chironomidae, namely the Podonominae (Brundin, 1966; matrix was prepared (but unpublished) and analysed under Cranston, Edward & Colless, 1987; Cranston & Edward, 1998), several criteria, but the results were used more to discuss Aphroteniinae (Brundin, 1966; Cranston & Edward, 1992) and parsimony as an objective criterion for analysis than to assess the Diamesinae, tribe Heptagyiae (Brundin, 1966). In all these relationships of taxa (Sæther, 1990a). In the same conference examples, the biotically-related landmasses comprise Australia, proceedings, Rossaro (1990) showed the results of a numerical southern South America and southern Africa (for the latter area, study of Orthocladiinae relationships, again without publishing the presence of an undescribed taxon of Heptagyiae is newly a matrix. These two efforts attempted to estimate the phylogeny noted here), with a New Zealand connection in certain groups. of the complete subfamily, or at least those taxa known in all life In the more speciose and cosmopolitan subfamilies stages. Such a remit is beyond the present study, and we seek Orthocladiinae and Chironominae, such austral relationships have the sister group for Botryocladius and the likely relationship of been little explored: Edwards (1931) denied their existence, in the genus with “similar” taxa. contrast to Freeman’s (1961) recognition of Australian / Patagonian-South Chilean links amongst some “more primitive Methods, materials and phylogenetics or apparently more primitive genera” of Chironominae and Orthocladiinae. Long-term studies in Australia, and more A variety of collection methods have been used. Pupae and recent collections from southern South America have revealed a their exuviae were collected by exposing one or more drift nets diversity of austral vicariant taxa, including several genera of with a 300 µm mesh to intersect flowing water surfaces for up to Orthocladiinae going beyond the “primitive” Stictocladius 24 h.. The association between larva, pupa and adult necessary Edwards (Freeman, 1961; Edward, 1989). One such genus for full taxonomic descriptions has been attempted by rearing appears to be phylogenetically relatively derived within the live larvae individually through to the adult. Live larvae were Orthocladiinae, and undescribed despite being speciose, and placed individually in a few ml of native (or tap) water in widespread and abundant in lotic and lentic in systems in cotton-wool stoppered 50mm x 10mm glass vials, and Australia and Patagonia/Chile. maintained at cooler than ambient temperatures, without Here we describe this taxon from Australia, Argentina and aeration. Any emerged adults and associated exuviae were Chile, provide diagnoses based on all stages (adult males and preserved in 70% ethanol. If metamorphosis did not occur within females, pupae and larvae), key all species based on pupal about one week, raising the temperature by about 5°C exuviae and most Australian species from adult males and sometimes hastened the process. larvae. Taxa based on pupae alone, without associated adults, Microscope slide preparation involved clearing where might be suspect as potential junior synonyms of described, necessary with 10% KOH, neutralisation and initiation of unreared, adult-based taxa. To guard against this possibility all dehydration with glacial acetic acid, then mounting from previously described orthocladiine taxa from Patagonia and propan-2-ol (isopropanol) into Euparal. Exuviae were displayed by dissecting the cephalothorax from the abdomen, and attempts R., river; R, R1, R4+5, wing veins R, R1 and R4+5 respectively, were made to remove pharate adults from exuviae. setal count; RM, radius to media crossvein; RO, ring organ, Morphological terminology follows Sæther (1980) and distance from base; S, south; S.Ch., sensilla chaetica; Scts, Cranston (1994) except where Langton’s (1994) suggested use scutellum, setal count; S.F., state forest; St.IX, sternite IX, setal of taenia (adjective taeniate) for “filamentous” or “lamelliform” count; Sq, squama, setal count; S.V., ratio of Fe + Ti : Ta1; TIX,

(LS) pupal setae is adopted. In addition, in pupal descriptions tergite IX. setal count; Ta 1-5, tarsomeres 1-5; temp., temporals, we refer to the conjunctive as if belonging to the segment setal count; th.h.l., thoracic horn length; Ti, tibia; U., upper; anterior to it, rather than the conventional but cumbersome vms.l., length of ventromental setae; V.R., ratio of length of Cu1 citation of the segment number that precedes and follows. : length of M; W, west; W.l., wing length (arculus to apex) in Many measurements of larvae and pupae are of exuviae but mm. larval length and head capsule measurements are based on complete fourth-instar larvae. Measurements of larval antennal Phylogenetics features and subsequent calculations of the antennal ratio are based on the sclerotised portions of the antenna, disregarding For phylogenetic analyses at generic level, taxa included the variably distended membranous intersegmental regions. are those suggested by female genitalic features (Sæther, 1977) Mensural features are tabulated to facilitate comparison. Unless to be close to Paralimnophyes, a genus to which Botryocladius indicated otherwise, measurements are in µm and rounded to shows some overall similarity, and the grouping to which some the nearest 5 µm except in cases where measurement at stages of Botryocladius will key. Other adult taxa with small maximum magnification provided accuracy to 1 µm. scalpellate acrostichals were included, as were two formally Many distribution data have been acquired from capture of undescribed taxa from Australia of putative generic rank drifting pupal exuviae. Geographical coordinates are cited to (Cranston, 1996) sharing some features with Botryocladius. Their nearest minute if map-derived, or to the second when taxon codes (“Son”) reflect only the discovery sequence (n) of GPS-derived. Locality data for Australia are cited in the “Sydney Orthoclads” (Cranston, 1996) and convey no following clockwise sequence: Northern Territory, Queensland, postulated phylogenetic relationship. All aforementioned taxa New South Wales, Victoria, Tasmania, South Australia, listed were scored for each life-history stage. Potentially-related north to south, followed by Western Australia, listed south to described taxa, e.g. Australian Kiefferophyes Freeman, are north. Unless specified otherwise, the collector is one or other excluded for lack of information concerning immature stages. or both of the authors, and repository is either Adult-only classification is rejected based upon empirical ANIC - Australian National Insect Collection, or evidence such as that of Sæther (1990a), who could not recover WAM (Western Australian Museum, under present care of a monophyletic Orthocladiinae, and Cranston & Edward (1998) junior author) for material from Western Australia. Other whose resolved Podonominae phylogeny collapsed for the repository abbreviations: BMNH - British Museum (Natural adult-only data partition. History) (now the Natural History Museum); FR - Dr F. Reiss, Characters scored are those believed a priori to be private collection; NMV - Museum of Victoria, Melbourne; potentially informative about relationships by virtue of their ZMBN - Museum of Zoological, Bergen, Norway; ZSM - variation within the selected taxa. Highly homoplasious Zoologische Staatssammlung München, Germany. characters from previous studies, and those with high intra-generic polymorphisms, generally are excluded; a few are Abbreviations treated as “?” as are a few unknowns most of which refer to female genitalic characters for taxa unavailable for coding. Data Morphological (adults unless stated) and geographic was analysed under parsimony with Hennig86 (Farris, 1988) abbreviations used in the text and tables: Ac, acrostichals; A.l., operating within a Microsoft Windows shell of Tree Gardener anal lobe setal number (pupa); Ant1-6, antennal segments 1-6 1.0 (Ramos, 1996) employing the option ie-; bb. Trees were length (larva); apn1-3, antepronotal seta 1-3 length (pupa); A.R., rooted with Diplocladius Kieffer as outgroup following its antennal ratio = length of ultimate flagellomere : combined unambiguous placement near the base of the Orthocladiinae by lengths of flagellomeres 1 to penultimate (adult) or length of Sæther (1990a) and Rossaro (1990). In initial analyses all basal segment : combined lengths of segments 2 to apex (larva); characters were treated as additive (ordered), followed by B.l, body length (thorax + abdomen of adult, otherwise total various permutations of non-additive treatments. Assessment of length of larva, pupa) in mm; Bl, antennal blade length (larva); confidence in some taxonomic results from the analyses were

B.V., combined length Fe + Ta + Ti1 : combined lengths ta2-4; Ck, made using T-PTP (Faith, 1991) tests, which compare the length creek; Clyp, clypeus, setal count; Cu1, length of vein Cu1;Dc, of minimal-length trees (mlts) against the lengths obtained by dorsocentral setal count (adult); dc1-4,, dorsocentral setae with similar searches of the data matrix in which within-character dc1-2 distance (pupa); E, east; Fe, femur; Fl (1-n), flagellomere randomisation has destroyed phylogenetically informative (1-n combined); fs.l, frontal seta length (pupa); GcIX, gonocoxite covariation (Faith & Cranston, 1991). Tests for robustness were IX, setal count; Gcx, gonocoxite length; Gst, gonostylus length; undertaken using T-PTP tests as implemented in PAUP 4.0.0d54 H.l., head capsule length, anterior labrum to posterior margin for Macintosh, with 100 randomisations of the data matrix. (larva); HV, hypopygium value = body length:gonostylus length Treating Botryocladius as a single terminal taxon doesn’t x 10; L, larva or Lake; L.aps., lateral antepronotals, setal count; allow assessment of internal relationships necessary to Le, larval exuviae; Le/Pe/m(f), reared adult male (female), with understand biogeography, and a second matrix was constructed associated larval and pupal exuviae; loc., locality; L.R., leg with species as terminals. Since much information is lacking ratio: tarsomere1 length: tibia length; M, length of vein M; M.w., concerning the adults and larvae and wishing to avoid filling a mentum width (compressed larval head capsule); Md, mandible matrix with “?” for unknown semaphoronts, we used the life length (larva); Mmw, median mental tooth width; ms.l, macroseta history stage that is fully represented, namely the pupa. Since length (pupa); N, north; N.P., national park; P, pupa; Pa, prealar the stage contains much informative variation in comparison with setal count; P1-3, Legs (1-fore, 2-mid, 3-hind); pc1-3, precorneal others, we assess the “pupal phylogeny” and analyse this data as seta 1-3 length (pupa); Pc.l (w), procercal seta length (width) above. (larva); Pe, pupal exuviae; Pm(f), pharate male (female) within pupa; P-orb, postorbital setal count; R, wing vein R, setal count; Botryocladius gen.n. paired median area; weak ventromental plate with strong, often branched, setae beneath; body with separate posterior and Type-species: Botryocladius grapeth sp.n., here designated. anterior parapods, weak to absent thoracic and abdominal setae, procercus always with 4 apical setae. Etymology: From botryus, Latin for a bunch of grapes, after The larva keys readily to Rheocricotopus in Cranston et al. the resemblance of the pupal thoracic horn to a bunch of grapes (1983), from which it can be distinguished by the relative lengths (see Fig. 6) (see also under B. grapeth), and cladius, Latin for a of antennal segments which decrease in size distally in diminutive branch (clade). Rheocricotopus, and in which there is never a filamentous 6th segment. The menta of certain formally undescribed Australian Diagnosis and recognition taxa resemble those of Botryocladius species (e.g. “MO5”, “SO2”, “SO3”; see Cranston, 1996). Notably the larva of “SO2”, Adults: Botryocladius adults have bare eyes with weak in which the fine 6th antennal segment and sparse ventromental dorsomedial extension; wings fully developed, with weakly beard was not drawn in Cranston (1996) and “SO3 species 2”, extended costa and sinuate Cu vein, finely punctate and lacking with an extremely similar antenna to Botryocladius, require macrotrichia on membrane, with setose squama; legs with strong inclusion in phylogenetic analyses on pupal and larval grounds. lateral spines on spur, with comb, without pulvilli; thorax with few hooked / scalpellate acrostichals in mid-thorax, without Generic description pleural setae; male hypopygium with strong virga and weak, bare anal point; female genitalia with large rectangular undivided Male. Small to medium sized, length 1.9-3.7 mm; wing length tergite IX, protruding setose gonapophysis IX, well developed 1.1-2.1 mm. Antenna with 13 flagellomeres, well-developed dorsomesal lobe, paired, ovoid, bare seminal capsules, curved/ plume extending to apex lacking strong subapical seta; groove looped spermathecal ducts. extending from flagellomere 3 or 4 to 13; sensilla chaetica on Botryocladius males key in Cranston et al. (1989) either to flagellomeres 2-5 and sub-apex of 13. A.R. 0.32-1.25. Head with Unniella Sæther or Paralimnophyes Brundin. Although eye bare with weak dorsomedial extension. Temporal setation sharing the mid-scutal scalpellate acrostichals with both, it is restricted to few small coronal setae and 3-7 linear postorbitals. precluded from either by the development of the anal point and Tentorium tapering at each end, swollen medium section virga, and from the latter by the weak punctation of the wing bearing indistinct sieve plate, cibarial pump rectangular with short membrane. Compared to the somewhat similar Compterosmittia cornua (Fig. 1B). Clypeus sparsely setose. Palps with 5 segments, Sæther, Botryocladius lacks the very extended costa and 5th >subequal 3-4, longer than 2nd, 3rd segment without sensilla pectinate gonostylar megaseta. Synorthocladius Thienemann chaetica. Thorax (Fig. 1C) uniform medium brown. resembles Botryocladius in many features, including the curved Antepronotum well developed, lobes not medially narrowed,

Cu1 and presence of a virga, differing mainly in R4+5 ending gaping, separated. Thoracic setation: 2-7 lateral antepronotals; distal of the apex of M3+4 in the latter genus, whereas it is proxi- 1-2, rarely up to 8, small hooked/scalpellate acrostichals in mal or above in Synorthocladius. The leg spur denticle devel- medium scutum; 5-9, rarely 13 uniserial dorsocentrals; 3-5 opment conveys no indication of relationship to the otherwise prealars; 4-9 uniserial scutellars. Wing (Fig. 1A) membrane with quite dissimilar Chaetocladius Kieffer and Bryophaenocladius fine punctation, without macrotrichia. R4+5 ending distal to M3+4. Thienemann. The female of Botryocladius exhibits character Brachiolum with 1 seta, R with 1-6, remaining veins without combinations that virtually preclude its keying in Sæther (1977), setae. Squama with 2-8 setae. Anal lobe moderately produced. but by circumventing some ambiguities in interpretation it can Costa with slight extension, FCu distal to r-m (V.R. 1.15-1.40), be keyed to a grouping around Limnophyes – Parorthocladius Cu1 curved, with recurved apex, faint before wing margin. Legs (couplets 61-2). with fore tibial spur very long, wider than tibial apex, medium Pupae: Pupae of Botryocladius have frontal setae (except B. tibia with two short, subequal spurs; hind tibia with one short, australoalpinus); unique botryoidal thoracic horn; dense one long spur, with comb (Fig. 1D); mid- and hind spurs weakly abdominal armament including long needle-like or triangular to strongly denticulate; pseudospurs absent. Sensilla chaetica spines on posterior of most tergites, including VIII; many tergal apparently absent. Pulvilli absent. Tergites unicolorous, with and sternal conjunctives spinose without hooklets; most L-setae scattered (disorganised) setation. Tergite IX with few apical semi-taeniate, anal lobe rounded/truncated, with 3 macrosetae setae. Anal point small, bare, apparently sometimes absent. and no fringe. Sternapodeme squared off, with weak to strong oral projections; Botryocladius pupae with needle-like spines key in Coffman phallapodeme well developed. Hypopygium (Figs 1E, 2A-F) with et al (1986) to Limnophyes and Paralimnophyes, whereas those virga present, variably developed, or perhaps occasionally lacking needle-like spines present irreconcilable character state absent; superior volsella rounded low contour or absent; conflicts. The genus Compterosmittia, not included in the inferior volsella subtriangular, rounded or with more or less above-mentioned key, somewhat resembles needle-spined elongated posterior projection, sometimes obviously double. Botryocladius (Cranston & Kitching, 1995). Several pupal taxa Gonostylus simple, with megaseta and small crista dorsalis. of Australian orthoclads share certain features with Botryocladius including “MO5”, which differs in having multiple prealars and Female. As for male, except body length 2.1-3.5 mm, wing dorsocentrals and spinose macrosetae (Cranston, 1996), Pirara length 1.1-1.8 mm. Antenna with 5 flagellomeres; sensilla Boothroyd & Cranston (1995) and “MO8” both of which have chaetica on all flagellomeres. A.R. 0.54-0.66. Head with 3-9 fine, short macrosetae and “SO1” which lacks any tergal postorbitals. Wing veins R, R1 and R4+5 setose; venarum ratio conjunctive spinule bands (Cranston, 1996). The most similar 1.2-1.4; squama with 2-8 setae. Genitalia (Fig. 2F,G,H) with of the formally undescribed pupal taxa are “SO2” and “SO3” in tergite IX large, rectangular, undivided, bearing 3-17 strong which the latter differs in lacking frontal setae and thoracic horn, submarginal setae; gonocoxite IX bulging, with 3-16 long and and the former differs mainly in having a non-botryose thoracic short setae; gonapophysis VIII divided, large ventrolateral lobe horn. overlying posterior part of weak dorsomesal lobe (that may Larvae: Botryocladius larvae have 5 or 6 segmented antenna represent no more than darker pigmented antero-lateral margin of moderate-length; 3rd antennal segment shorter than 4th; bifid of vaginal cavity); apodeme lobe intra-specifically variable, SI seta; elongate simple SII seta; mentum with domed or broadly slender, line-like, weakly curved to interrupted medially; notum strong, extending further anterior than seminal capsules; 2 developed conjunctival spine bands on (III) IV-VII. Anal lobe seminal capsules pale, ovoid to pear-shaped, without somewhat elongate, with antero-median shagreen, with 3, microtrichia, with well developed neck; spermathecal ducts exceptionally 4, subequal stout macrosetae, ranging in length either with one loop, or with several bends, either with from slightly shorter than anal lobe to appreciably longer, pronounced bulbs before (? common) opening on spermathecal usually apically hooked. Male genital sac extends posteriorly eminence, or apparently without bulbs and with separate beyond anal lobe; that of female much shorter. Setation. openings; labia simple and weakly sclerotised. Tergite X and Segment I with 5 D, 1-2, exceptionally 3 L and 2-4 V setae; cerci small, postgenital plate not evident. segments II-VII with 5 or exceptionally 9-10 D (Fig. 3E), 4 strong and simple to semi-taeniate L, and 5 V setae; segment VIII with Pupa. Small to medium-sized, length 2.1-4.2 mm. Exuviae 1-2 D, 3-4 L and 1-2 V setae. pale to medium brown, cephalothorax usually more pigmented than abdomen, thoracic horn pale to dark brown, apophyses Larva. Fourth-instar larva medium-sized, 3.1-5.8 mm long. often dark brown. Cephalothorax: frontal setae on frons, often Antenna (Fig. 8A) either 5 or 6-segmented, with 6th, when conspicuous, elongate and arising from short tubercles, absent present, very fine filament, segment 3 subequal to or more in B. australoalpinus. Frontal apotome smooth to rugulose, with usually shorter than segment 4. Antennal Ratio 0.72-1.7. Ring frontal warts (Fig. 3B) in one species. Ocular field with one organ near base of segment 1; antennal blade simple, extending vertical seta. Thorax with 2 median and one lateral antepronotals, to apex of segment 3, in one species to apex of 5th; Lauterborn

3 precorneals, pc1 and pc2 long, pc3 shorter, in a variety of organs varying from distinct and subequal in length to antennal arrangements, from linear to triangular; 4 dorsocentrals in 2 pairs. segment 3, to somewhat weaker; style very short, perhaps Thoracic horn (Fig. 6) distinctive, elongate ovoid, variably sometimes absent. Labrum (Figs 8I, 10) with SI bifid, SII-IV covered with rounded tubercles, from 60 - 215 µm long. simple, SII thickened and retracted posteriorly to lie external to Dorsum of thorax variably rugulose. Prealar area crenulate/ fine SIII, with SIVa externally on similar alignment; one chaeta rounded, usually with 2 setae, sometimes with one, 3 or 4. Wing serrate, remainder simple; spinulae strong. Premandible with one sheath without pearls, sometimes anteriorly darkened and or 2 apical teeth and moderately developed premandibular brush. crenulate. Abdomen: pedes spurii A and B absent. Tergites (Figs Pecten epipharyngis of 3 unfused scales. Ungula short and squat 3A, 4, 5): I without spinules / shagreen; II with anterior shagreen, with few short and simple or pectinate chaetae. Basal sclerite with or without posterior transverse band of elongate spinules, spatulate. Mandible (Fig. 8C, E, G, H) with outer and inner without hook row, with or without transverse uni- to multiserial margins smooth. Length of apical tooth either shorter than 1st conjunctival spine band; III with widespread shagreen of spinules inner tooth and displaced onto different plane, subequal to com- that may be larger than on other tergites, with posterior bined width of 4 inner teeth, or on same plane and slightly shorter transverse band of weakly to strongly developed spines, than combined width of 3 inner teeth. Seta subdentalis present, conjunctive with transverse multiserial band of anteriorly directed more or less rounded. Seta interna with 5-7 simple branches. spinules or spines, sometimes medially interrupted; IV with Mentum (Figs. 8B, D, F, 9) with median tooth either apparently anterior shagreen less dense than on III, with posterior simple and broad, or weakly to strongly paired with variably transverse row of broad spines, sometimes medially divided and developed nipple on each median tooth; 5 pairs of lateral teeth situated more anteriorly, sometimes partially or completely either decreasing on even slope, or with outermost tooth more developed as somewhat mace-like structure, conjunctive with aligned with its inner neighbour. Ventromental plate (Fig. 9) transverse multiserial band of anteriorly directed fine spines, often narrow or may extend to outermost mental tooth (extent depends very dense medially; V and VI with weaker anterior shagreen, on degree of compression); beard of sparse to strong setae which posterior transverse band of spines and conjunctive with dense may be quite broad and apically bifurcate. Maxilla (Fig. 8J) squat, spinules or fine spines; VII and VIII with anterior shagreen and bearing few simple setae. Body without any evidence of lateral posterior transverse band of spines, conjunctives bare. Spines setae. Anterior parapods separate, with crown of elongate, of posterior bands on tergites may be either broad, triangular, simple, pale spines and yellow, pectinate claws, merging into and usually less than 50 µm long, or narrow, needle-like and more basal area of fine, pale, straight spines almost linking 2 greater than 50 µm long. Sternites (Figs 3F, 7): I and usually II parapods at base. Posterior parapods separate, with apical group bare, III-VIII may have anterior and posterior shagreen, with of simple claws. Procercus as wide as high, sometimes partly posterior band developed as transverse spine band on pigmented, bearing 2 very proximate median setae and 4 successively more posterior segments, with sexual dimorphism medium-length anal setae. Two pairs of elongate-globular anal in expression of sternite VIII band in two species; variably tubules.

Keys to Botryocladius species

Males (of Australian species only)

1. Virga minute (<5 µm) (Fig. 2E), or absent petrophilus sp.n. - Virga at least 15 µm long (Fig. 2A-C, F) 2

2 A.R. <0.4; inferior volsella rectangular (Fig. 2F). Patagonian edwardsi sp.n. - A.R. >0.45; inferior volsella variably rounded (Fig. 2A-E). Australian 3

3 Dorsocentrals more than 10; inferior volsella clearly comprising 2 parts, postero-ventral part protruding (Fig. 2D) collessi sp.n. - Dorsocentrals fewer than 10; inferior volsella not obviously comprising 2 parts, postero-ventral part insignificant 4

4 Dorsal gonocoxal surface densely microtrichiose to base (Fig. 2B); volsella without extensive bare lobe brindabella sp.n. - Dorsal gonocoxal surface bare in basal half (Fig. 2A) or with few microtrichia (Fig. 2C); volsella with variably developed bare lobe 5 5 Basal gonocoxite with strongly bulging inner contour; dorsal gonocoxal surface with few microtrichia (Fig. 2C) bibulmun sp.n. - Basal gonocoxite with smoothly curved inner contour; dorsal gonocoxite surface bare in basal half (Fig. 2A) grapeth sp.n.

Pupae

1. Tergal armament includes elongate parallel-sided, needle-like, fine spines at least on tergite III (Figs 3E, 4, 5C) 2 - Tergal armament includes broad-based, triangular spines but lacks elongate, needle-like spines (Figs 3A,D, 5A,B) 6

2 Tergite II conjunctive without spines (Fig. 5C) 3 - Tergite II conjunctive with multiserial spines (Fig. 4) 4

3 Tergite IV with identical armament pattern to tergites III and V, with 5D setae (Fig. 5C); exuviae pale yellow, thoracic horn with few apical tubercles (Fig. 6J) mapuche sp.n. - Tergite IV with very reduced antero-median armament of spinules compared to tergites III and V, with 7-10D setae (Fig. 3E); exuviae brown, thoracic horn tuberculose (Fig. 6K) tronador sp.n.

4 Tergite IV with posterior spine band medio-anteriorly directed and interrupted, but without development as mace (Fig. 4A) brindabella sp.n. - Tergite IV posterior spine band developed as mace-like spinose clump (Figs 3C, 4B,C) 5

5 Tergite IV with mace-like structure at median end of linear spine row (Fig. 4B) collessi sp.n. - Tergite IV with spines restricted to mace-like structure (Fig. 4C) mdfrc sp.n.

6 Cephalic area and pedicel sheaths with warty protuberances (Fig. 3B) tasmania sp.n. Cephalic area and pedicel sheaths without warts 7

7 Two precorneal setae long and semi-taeniate, pc3 thin and short (Fig. 6I); posterior spines of tergite VIII very strong (>50 µm long) glacialis sp.n. - Precorneal setae non-taeniate, shorter, and less divergent in length; posterior spines of tergite VIII weaker (<40 µm long). 8

8 Tergal conjunctive II bare (Fig. 3A) 9 - Tergal conjunctive II strongly spinose (Fig. 5A) 11

9 Thoracic horn expanded from base to give somewhat triangular shape, without distinct neck (Fig. 6G); spinose tergal conjunctives (III-VI) with sparse, irregularly bi- to triserial spines; posterior bands of spines virtually uniserial, spines and spinules pale freemani sp.n. - Thoracic horn with distinct neck, apically globular (Fig. 6A,D); spinose tergal conjunctives (III-VI) with dense, clearly multiserial spines; posterior bands of spines more or less biserial, spines and spinules brown 10

10 Frontal setae present; dorsal setae D1-D4 not aligned (Fig. 3A); sternal shagreen comprising spinulose conjunctives IV-VI and posterior transverse bands of spinules on sternites III-VIII (Fig. 3F) grapeth sp.n. - Frontal setae absent; dorsal setae D1-D4 on antero-posterior alignment (Fig. 3D); sternal spinules restricted to conjunctives IV-VII (Fig. 7E) australoalpinus sp.n.

11 Tergite II with dense shagreen extending across much of tergite, connecting to posterior transverse row of strong spines (Fig. 5A); male sternite VIII with posteromedian cluster of spines (Fig. 7I) edwardsi sp.n. - Tergite II with sparse shagreen, dissociated from any posterior row, which if present is of weak tubercles; male sternite VIII lacking posterior spines 12

12 Thoracic horn >100 µm long, with neck broad, indistinct (Fig. 6F); sternal conjunctive III with transverse band of spinules (Fig. 7G); apex of anal lobe conventionally developed (Fig. 3H) petrophilus sp.n. - Thoracic horn <100 µm long, with narrow, distinct neck (Fig. 6E); sternal conjunctive III bare (Fig. 7F); apex of anal lobe with toothed ridge at bases of anal macrosetae (Fig. 3G) bibulmun sp.n.

Larvae (of Australian species only)

1 Median mental tooth single, domed; ventromental plate broad, extending towards outer margin of mentum (Figs 8B, 9C); mandible with distinct 4th inner tooth, as large or larger than other inner teeth (Fig. 8C) freemani sp.n. - Median mentum with paired teeth (Fig. 8D,F) (though difficult to discern in worn specimens); ventromental plate thin, not extending to outer mentum; mandible with 3 distinct inner teeth, 4th absent (Fig. 8E), indistinct or smaller than other inner teeth (Fig. 8G) 2

2 Outermost mental tooth outside line of slope of mentum, outermost two teeth subequal (Fig. 9A,B); premandible with one tooth; mandible with three inner teeth, and no indication of 4th (Fig. 8E) 3 - Outermost mental tooth on line of slope of mentum; outermost tooth smaller than its neighbour (Fig. 9C-E); premandible with two teeth (Fig. 8I); mandible with 4th inner tooth variably indicated (Fig. 8G) 4 3 Beard of mentum with coarse, serrate setae (Fig. 9A) grapeth sp.n. - Beard of mentum with finer, basically simpler setae (Fig. 9B) bibulmun sp.n.

4 Median mental teeth rounded; beard of strong, branched setae extending lateral to outermost mental teeth; ventromental plate weak, linear (Fig. 9D) petrophilus sp.n. - Median mental teeth each with nipple (Fig. 8F); beard of weak, short simple setae not extending beyond outermost mental teeth; ventromental plate stronger, bulging (Fig. 9E) brindabella sp.n.

Etymology. This species, the type of the genus, has been Botryocladius grapeth sp.n. known colloquially as “grape th” since its first discovery and inclusion in identification manuals (e.g. Cranston, 1996), based “Grape th species A”, Cranston, 1996. on the fanciful resemblance of the pupal thoracic horn (“th”) to a bunch of grapes. In response to pleas from users not to alter Material examined. Holotype Le/Pe/m, AUSTRALIA: New the name, this species is named B. grapeth, to be treated as a South Wales, 34º38’S 150º33’E, Belmore Falls, Barrengarry R., noun in apposition, and to be pronounced “grape tee aitch”. 16.xii.1992 (Cranston) (ANIC). Paratypes, New South Wales: Male. Abdomen and thorax uniformly medium brown, with 2L, 2Pe, 1Pf, 34º38’S 150º33’E, Belmore Falls, Barrengarry R., vittae scarcely darker; legs pale brown. Hypopygium (Fig. 2A) 3.iv.1991; 2Le/Pe/f (1 to ZMB), 1L/P, 1Pe, same loc., 16.xii.1992; with well developed virga, anal point absent to small, tergite IX 1Pe, 34º47’S 150º30’E, Bungonia, Bungonia Falls, 11.xi.1988; with 0 or 1 seta, gonocoxite with substantially b are volsella. 4Pe, 35º05’S 150º08’E, 6km NE Nerriga, Endrick R., 1.ix.1988; Mensural features as in Table 1. 1L, 1L(P), 35º22’S 148º42’E, Brindabella, Bramina Ck, Female. As male, except in generic antennal and genitalic 29.viii.1988; 8Pe, 35º16’S 150º03’E, Morton N.P., Wog Wog (Fig. 2G,H) features. Mensural features as in Table 2. Ck, 25.iv.1994; Pe, 35º21’S 149º44’E, Warri Bridge, Shoalhaven Pupa. Pale brown, apophyses medium brown, thoracic horn R., 15.iii.1992; Pm, 36º24’S 148º40’E, Jindabyne, Rushs Ck, (Fig. 6A) medium brown. Abdominal spines and spinules darker 12.xii.1987; Pe, 35º34’S 150º02’E, Currowan S.F., Cabbage Tree brown than cuticle. Frons, anteromedian thorax and prealar Ck, 30.iii.1994; Pe, 35º23’S 149º55’E, Monga S.F., Mongarlowe projection rugulose. Tergites as in Fig. 3A, sternites as in Fig. R., 2.ii.1991; Le/Pm, Le/Pe/f, 35º35’S 149º28’E, Molonglo R., 3F. Mensural features as in Table 3. above Captains Flat, 12.ii.1989; 2Pe, same loc., 7.iii.1989; Pf, Larva. Head capsule yellow, with dark brown mentum, Captains Flat, Molonglo R., 30.i.1988 (Atkins); 1Le/Pm, 2Pe, apical 2/3 of mandible and premandible dark brown. Antenna 36º36’S 149º25’E, Brown Mt., Rutherford Ck, 17.xii.1990; 2Pe, yellow. Occipital margin pale. Premandible with one apical tooth, 35º38’S 148º34’E, Kosciusko N.P., Murrumbidgee R., 13- mandible with 3 inner teeth and darkened mola. Mentum (Fig. 14.xi.1993; 2Pe, 35º39’S 148º28’E, Kosciusko N.P., 8D) with paired median teeth, often worn to appear single, with Yarrangobilly, Yarrangobilly R., 14-15.i.1991. Australian Capi- outermost tooth somewhat blunt and lying anterior to line of tal Territory: 7Pe, 35º27’S 148º57’E, Tidbinbilla, Tidbinbilla Ck, even slope of mentum. Ventromental plate weak, beard of 5-7 19.ii.1989; Le/Pe/m, L(P), 35º31’S 148º56’E, Corin, Gibraltar stout, apically branched setae (Fig. 9A). Procercus brown. Falls, 13.x.1993, Le/Pe/f, Le/Pe/m, same loc., 23.x.1993, Le/ Anterior and posterior claws pale golden. Mensural features as Pe/m, same loc., 23.xi.1993, 2Pe, same loc., 24.x.1991 in Table 4. (Drayson); 1Pe, same loc., 15.ii.1997. Victoria: 3P, 1Pe/m, 1Pm, Comments. Botryocladius grapeth is the commonest 36º32’S 147º25’E, Mitta R., 18.v.1990 (MDFRC); 3Pm (1 to species of the genus in south-eastern Australia. As with all ZMB), 29Pe (2 to ZSM, 2 to BMNH, 1 to NMV), 36º48’S Botryocladius, identification of the unreared adult is not easy. 146º51’E, Buckland R., 6.v.1991 (Cook, Cranston & Nielsen), The male may be distinguished by the rather few microtrichia on Pm, same loc., 6.xi.1990 (Cook); Pe, same loc., 6.xi.1990 (Cook, the otherwise bare basal 2/3 of the ventral surface of the Cranston & Nielsen), 1Pe/m, same loc., 6.v.1991, ex-wood; Pe, gonocoxite, which has a rather smoothly curved inner contour. 36º54’S 147º27’E, U. Tambo R., “CC11”, 23.iii.1990 (Hortle); No distinguishing features have been found for the female. 3Pe, 36º54’S 147º27’E, Omeo Hwy, Big R., 26.i.1989; P, Pm, The pupa of B. grapeth lacks needle-like spines, and amongst 37º00’S 147º52’E, Tambo R., up Wilda Weir, 30.iii.1993 this grouping can be distinguished by tergite II having (Hortle); Pe, 37º28’S 145º45’E, Steavenson R., 20.iii.1992 reasonably strong shagreen but with the conjunctive bare. From (Downes) (NMV). Tasmania: 2Pe, 41º04’S 147º15’E, The Bot- the species with otherwise similar tergites, B. grapeth can be tleneck, Ansons R. 22.v.1993; 1Pe, 41º10’S 148º54’E, nr distinguished from B. tasmania by the lack of rugose frontal Weldborough, Weld R., 20-25.ii.1993 (Trueman & Cranston); warts and from B. freemani by the globular-shaped thoracic horn. 2Pe, same loc., 23-24.viii.1993; 11Pe, L. Sorrell, 18.iii.1997, The larva of B. grapeth has a double median mental tooth, 810 m.a.s.l. (Wright). 41º04’S 147º15’E,. South Australia: 2Pe, and the outermost tooth subequal in size to its inner neighbour, 35º57’S 136º42’E, Kangaroo I., Rocky R. (mid), 29-30.ix.1994; and lying somewhat lateral to it, rather than on the line of slope 1 Pe, 35º57’S 136º40’E, Kangaroo I., Rocky R. (mouth), of the mentum. Botryocladius bibulmun, a western Australian 1.x.1994. Western Australia: Le/Pe/f, 34º22’S 118o14’E, Stir- species with a larval mentum resembling that of B. grapeth, ling Range, differs in having virtually unbranched setae in the beard (Fig. Bluff Knoll, 20.xi.1990. 9B, compare with Fig. 9A). Excluded from type series: Queensland: 6Pe, 18º13.1”S This species is common in south-eastern Australia, and the 14548.5”E, Goddard Bridge #11, 9/10.vi.1997 (McKie), Pe, finding of several exuviae and 1 larva from tropical North 26º03.00”S 153º04.29”E, Cooloola N.P., Frankis Gulch, Queensland is somewhat anomalous. The exuviae, similar to the 6.iv.1996; New South Wales: 6Pe, 3L, 36º24’16”S 148º18’41”E, type except for lacking transverse bands of spines on sternite III, Kosciuszko N.P., Blue L., 1875 m. a.s.l., 3.ii.1988 (Cranston & are excluded from the type series. Such a finding of apparently Dimitriadis). Tasmania: 2Pe, 41º54’S 146º30’E, Central Plateau, cool-water taxa in streams of North Queensland is unexceptional L. Kay, 1150 m. a.s.l., 14.iii.1997 (Wright); 2Pe, 42º07’S - although water temperatures tend to be higher (at low 20’sºC), 147º10’E, L. Sorrell, 810 m. a.s.l, 18.iii.1997 (Wright); 1Pe, 42º stream shading seems to permit persistence of cool temperate 54’S 146º28’E, L. Ada, 16.iii.1997 (Wright). Gondwanan taxa (e.g. Stictocladius, Riethia, “MO5” etc). Although virtually all records are from cool, shaded, the Murray Darling Freshwater Research Centre (MDFRC) for running waters several pupal exuviae seemingly identical with many contributions to our understanding of the taxonomy of this species have been recovered from high altitude lakes with freshwater invertebrates in general, and Chironomidae in wave-swept shores. At Blue Lake, one of only three (Pleistocene) particular. To be treated as a noun in apposition and pronounced “glacial” tarns on mainland Australia, several larvae identical to as the reader wishes. those of B. grapeth were found on a mossy/algal layer on large Adult. Unknown. rocks exposed to wave action, but probably at the precise air/ Pupa. Very pale yellow, apophyses pale brown, thoracic horn water boundary under still conditions. very pale yellow, similar to B. collessi (Fig. 6B). Abdominal spines and spinules pale to golden yellow. Frons, anteromedian Botryocladius brindabella sp.n. thorax and prealar projection weakly rugulose. Tergites as in Fig. 4C, sternites as in Fig. 7B. Mensural features as in Table 3. “Grape th species B”, Cranston, 1996 Larva. Unknown. Comments. Botryocladius mdfrc, from which only the pupal Material examined. Holotype Le/Pe/m, AUSTRALIA: exuviae is known, belongs with a coherent group of taxa with Australian Capital Territory, 35º25’S 148º30’E, Brindabella long, needle-like tergal spines and with conjunctive II with many, Ranges, Moonlight Hollow, 20.x.1991 (Cranston). Paratypes, multiserial spines. As in B. collessi, posterior spine band on tergite Pm, 35º22’S 148º51’E, Brindabella Ranges, Lees Ck, 2.xi.1989 IV is modified, but the mace-like structure (Figs 3C, 4C) is very (Cranston); same loc., ex-wood, 8.xi.1995 (McKie); Pe, 35º31’S much more developed in B. mdfrc, and there are few or no strong 148º56’E, Corin, Gibraltar Falls, 11.vii.1989. spines lateral to this structure. Confirmation of the species Etymology. Named for the range of mountains to which this distinction comes from the sternal armament patterns, in which species appears to be restricted. To be treated as a noun in the shagreen is very much weaker, especially on sternite III in B. apposition. mdfrc (Fig. 7B) compared to B. collessi (Fig. 7C). Male. Abdomen and thorax uniformly golden brown, with vittae no darker; legs pale. Hypopygium (Fig. 2B) with well Botryocladius collessi sp.n developed virga, anal point about 10 µm long, tergite IX with 2 or 3 fine setae, and gonocoxite with very microtrichiose dorsal Material examined. Holotype 1Pm, AUSTRALIA: New surface, volsella right-angled rather than lobed. Mensural South Wales: 36º36’S 149º47’E, Brown Mt., Rutherford Ck, features as in Table 1. 2.x.1990 (Cranston). Paratypes, 3Pe, as holotype except Female. Unknown. 16.x.1990 (Cranston & Edward). Pupa. Very pale yellow, apophyses pale to medium brown, Etymology. Botryocladius collessi is named for our colleague thoracic horn pale yellow, similar to B. collessi (Fig. 6B). Don Colless, who first recognised the Gondwanan significance Abdominal spines and spinules pale golden yellow. Frons, of the Rutherford Creek type locality, confirmed by the anteromedian thorax and prealar projection weakly rugulose. collections there by Lars Brundin. Sadly recent forestry Tergites as in Fig. 4A, sternites as in Fig. 7A. Mensural features practices in the area have damaged this famous temperate as in Table 3. Gondwanan locality. Larva. Head capsule pale yellow, with medium brown Male. Teneral abdomen and thorax pale brown, with vittae mentum and apical 1/2 of mandible. Antenna pale yellow, with no darker; legs pale. Hypopygium (Fig. 2D) with well Lauterborn organ as long as segment 3. Occipital margin pale. developed virga, anal point about 25 µm long, tergite IX with 4 Premandible golden, with 2 apical teeth; mandible with 3 inner fine setae, and gonocoxite with bilobed volsella. Mensural teeth and darkened mola (Fig. 8G). Mentum (Fig. 8F) with paired features as in Table 1. median teeth, each with median nipple, with outermost tooth Female. Unknown. pointed and lying on line of even slope of mentum. Ventromental Pupa. Pale yellow, apophyses pale to medium brown, plate narrow, elongate, with beard of 6 short, simple setae (Fig. thoracic horn (Fig. 6B) yellow. Abdominal spines and spinules 9E). Anterior and posterior claws pale golden. Procercus pale. pale to golden yellow. Frons, anteromedian thorax and prealar Mensural features as in Table 4. projection rugulose. Tergites as in Fig. 4B, sternites as in Fig. Comments. The male of B. brindabella has a well developed 7C. Mensural features as in Table 3. virga, and uniquely, an extremely microtrichiose dorsal Larva. Unknown. gonocoxite surface (Fig. 2B). The pupa of B. brindabella Comments. The male of B. collessi, which is known only belongs with a coherent group with long, needle-like tergal spines from a pharate specimen, may be distinguished from the other and with conjunctive II with many, multiserial spines. The spines known Australian males by the high number of dorsocentral of the posterior band on tergite IV are somewhat modified, but setae, and the bilobed nature of the inferior volsella (Fig. 2D). not as much as in B. collessi and B. mdfrc (there is no evidence For distinguishing the pupa, see above under the similar of a clustering of spines at the median division). The larva has a species B. mdfrc. distinctive mentum, with a median nipple on each of the paired median mental teeth, and the beard setae are simple and short, Botryocladius tasmania sp.n. lying under a distinctive ventromental plate. Material examined. Holotype Pe, AUSTRALIA: Tasmania, Botryocladius mdfrc sp.n 41º10’S 147º54’E, N.E. Tasmania, nr Weldborough, Weld R., 12-17.xi.1993 (Trueman & Lee). “Grape th species C”, Cranston, 1996 Paratypes, 3Pe, as holotype; 5Pe, same loc., 20-21.ii.1993. Etymology. The species is named after the island state of Material examined. Holotype Pe, AUSTRALIA: 36º48’S Tasmania, the only currently known location. 146º51’E, Buckland R., 6.v.1991 (Cook, Cranston and Nielsen). Adult. Unknown. Paratypes, 2Pe, as holotype. Pe, 36º07’’S 146º54’E, New South Pupa. Cephalothorax pale brown, abdomen darker golden Wales: Aldbury, Murray R., 31.vii.1991 (Cook); Pe, Victoria, brown with strongly pigmented apophyses and thoracic horn (Fig. Pigs Point, Mitta Mitta R., 3.xii.1991 (MDFRC). 6C) medium brown, darker than cephalothorax. Abdominal Etymology. This species is named B. mdfrc to acknowledge spines and spinules darker brown than cuticle. Frons (Fig. 3B) with strongly rugulose warts, anteromedian thorax and prealar 34º51.57”S 116º25.17”E, Shannon N.P., Forth R., 30.x.1997; projection rugulose. Tergites similar to B. grapeth (Fig. 3A) 34º50’S 116o33’E Le/Pf, Deep R., 5.xii.1994 (ANIC); Pe, except with conjunctival bands with 8-10 irregular rows of 34º40’S 116º23’E, Shannon N.P., Fish Ck, 24.xi.1994 (ANIC); stronger anteriorly-directed spinules, instead of 5-6 irregular 4Pe, 34º39’S 116o22’E, mid-Shannon R., 23-24.xi.1994 (ANIC); rows. Sternites as in Fig. 7D. 1Pm, 34º30’S 115o58’E, nr Pemberton, Warren R., .ii.1994 Larva. Unknown. (ANIC); 3Pe, 32º25’S 115º47’E, Carey Bridge, Carey Ck., Comments. Botryocladius tasmania is known only from the 23.xi.1994 (ANIC); Pe/m, 34º24.47"S 115o52.29"E, Beedelup pupal exuviae, which resembles that of B. grapeth, differing Brook, Steep Rd., 15.x.1994; 2Pe, 34º13’S 115º46’E, Barlee particularly in the presence of rugulose warts on the cephalic Ck, Dickson Rd crossing, 25.xi.1994 (ANIC); Le/Pe/m, 32º42’S area and antennal bases. The strong development of posterior 115º40’E, Ludlow R., Claymore Rd, 6.viii.1994; Le/Pe/f, 32º15’S spines and conjunctival spinules on sternite III also aids in 116º04’E, Seldom Seen Brook, 6.x.1983; 4L, 32º14’S 116º04’E, differentiation from the otherwise potentially sympatric B. Waterfall Gully, 15.vi.1983 (Bunn); Le/P, 32º08’S 116o06’E, grapeth. Canning R., 26.xi.1978, Pe/m, same loc., 6.xii.1978, 10L, 2L/P, 3P, Pe/f, same loc., 21-22.iii.1989, 2L(P), same loc., 24.viii.1989; Botryocladius australoalpinus sp.n. Pm, 32º03’S 116º03’E, Lower Canning R., 20.ix.1985. Etymology. Named for aboriginal inhabitants of the south- Material examined. Holotype Pe, AUSTRALIA: Tasmania, west of Western Australia and their migratory track that traverses 1Pe, 41º47’S 146º16’E, King Solomons Jewels pond #2, much of the range of this taxon. To be treated as a noun in appo 17.iii.1997, 1185 m. asl, (Wright). Paratypes: 1Pe, as holotype; sition. 9Pe, 41º38’S 145º57’E, Cradle Mt N.P., L. Dove, 21.iii.1997 Male. Abdomen and thorax brown, with vittae no darker; (Wright), 1Pe, 42º06’S 146º07’E, Forgotten L., 25.iii.1997, 980 legs medium brown. Hypopygium (Fig. 2C) with well m.asl., (Wright); 1Pe, 42º06’S 146º08’E, Shadow L., 19.iii.1997, developed virga and anal point about 25 µm long, tergite IX 950 m. asl, (Wright); 2Pe, 42º42’S 146º35’E, Mt Field N.P., with 2 fine setae, and gonocoxite with well developed bare Platypus Tarn, 11.iii.1997; Pe, 42º43’S 146º35’E, Mt Field N.P., volsella. Mensural features as in Table 1. Dobson Tarn, 12.iii.1997 (Wright). Female. As male, except in generic antennal and genitalic Etymology. From Australo - adj for Australia, and alpinus - features. Mensural features as in Table 2. for the alpine landscape in which the species occurs. Pupa. Pale yellow-brown, apophyses weakly indicated, Adult. Unknown. thoracic horn (Fig. 6E) golden brown. Abdominal spines and Pupa. Pale brown, apophyses medium brown, thoracic horn spinules golden brown. Frons, anteromedian thorax and prealar (Fig. 6D) dark grey-brown. Frontal seta absent. Abdominal projection weakly rugulose. Apex of anal lobe, bases of spines and spinules darker brown than cuticle. Frons, macrosetae, unconventionally developed, crenulate (Fig. 3G). anteromedian thorax and prealar projection rugulose. Tergal Tergites as in B. edwardsi (Fig. 5A) except for tergite II having armament similar to B. grapeth (Fig. 3A), except spines stronger very weak shagreen; sternites as in Fig. 7F. Mensural features as on posterior transverse band, almost needle-like on posterior in Table 3. tergites, and D1-4 more or linearly aligned (Fig. 3D); sternites Larva. Head capsule golden yellow, with dark brown as in Fig. 7E. Mensural features as in Table 3. mentum and apical 2/3 of mandible. Antenna yellow, with Larva. Unknown. Lauterborn organ as long as segment 3. Occipital margin golden, Comments. The pupa of B. australoalpinus can be slightly darker than head capsule. Premandible brown with one distinguished by the lack of frontal setae. B. austroalpinus apical tooth. Mandible with three inner teeth and darkened mola. resembles B. grapeth having tergite II conjunctive bare, but it Mentum (Fig. 8D) with paired median teeth, often worn to differs in the black globular thoracic horn contrasting with the appear as single, with outermost tooth blunt and lying anterior yellow thoracic background colour, and by the greater strength to line of even slope of mentum. Ventromental plate broader, of the long spines. The transverse spine bands on more posterior with beard of 8-10 long, broad, barely branched setae (Fig. 9B). tergites have very long spines that approach needle-like. Anterior and posterior claws golden yellow. Procercus pale Furthermore this species differs from B. grapeth and all other brown. Mensural features as in Table 4. congeners in the ventral shagreen pattern, with spinules restricted Comments. Botryocladius bibulmun is the common to transverse conjunctival bands on IV-VII, without any sternal Botryocladius species in cooler, shaded, running waters of spinules. In the short and stout, anal macrosetal shape, only B. western Australia. The male may be distinguished on the petrophile amongst Australian Botryocladius species approaches genitalia by the rather few microtrichia on ventral surface of the B. australoalpinus. The closest species on sternal (and gonocoxite, which has a distinctive strongly developed lobe on macrosetal) features is the Patagonian B. mapuche, which has the medio-basal contour. short macrosetae and ventral armament virtually restricted to The pupa of B. bibulmun lacks needle-like spines, and can conjunctives IV-VII. be distinguished amongst this grouping by tergite II having a This species appears to be restricted to high elevation tarns spinose conjunctive. From Western Australian B. petrophilus, in Tasmania, with no records from comparable mainland alpine otherwise sharing these features, the possession of a narrow, lakes. Although B. grapeth occurs in both mainland and distinct neck to the thoracic horn and bare sternal conjunctive Tasmanian high altitude lakes, in no situation do both species III allow separation. The unusual structure at the anal lobe apex co-occur. (Fig. 3G) is shared only by the Patagonian B. glacialis. The larva of B. bibulmun has a double median tooth to the Botryocladius bibulmun sp.n. mentum, and the outermost tooth subequal in size to its inner neighbour, and lying lateral to it, rather than on the line of slope Material examined. Holotype Le/Pe/m, AUSTRALIA: south- of the mentum. Botryocladius grapeth an eastern Australian west Western Australia, 32º30’S 116º02’E, Foster Brook. species with a larval mentum resembling that of B. bibulmun, 2.ix.1982 (Edward). differs in having all beard setae branched (Fig. 9A, compare Paratypes Le/Pe/m (ANIC), Le/Pe/f, as holotype; 2Pe, with Fig. 9B). 34º52’S 117o16’E, Denmark R., 3.xii.1994 (ANIC); Le/Pe/m, Botryocladius petrophilus sp.n. loc., 24.vii.1990; L, same loc., 11.x.1990; L, 32º08.25"S 116º10.38"E, Death Adder Ck, 6.viii.1985. Material examined. Holotype Pm, AUSTRALIA: Northern Excluded from type series: 2L, New South Wales, 33º37’S Territory: 25º20’S 130º43’E, Kata Tjuta [Olgas], Valley of Winds, 150º42E, Nepean R., Yarramundi, .iv.1996 (AWT); L, 5.vi.1992 (Cranston) (ANIC). Paratypes, m, f, Pf, 8Pe, 3L, as Australian Capital Territory, 35º22’S 148º50’E, Brindabellas, holotype. New South Wales: Le/Pm, 7Pe, 34º05’S 146º12’E, Blundell’s Ck., 30.iv.1998. Cocoparra N.P., Woolshed Ck, 31.x.1993 (Cranston). Western Etymology. Named for Dr Paul Freeman, erstwhile Keeper Australia (all. coll. Edward): Pe/m, 32º41’S 119º21’E, Hyden, of Entomology at the Natural History Museum, London, and Lily McCarthy Rock, 16.xii.1992; 3L, 32º19’S 119o09’E, King reviser of the Australian Chironomidae fauna. Rocks, 7.vii.1990; Le/Pe/m, Pe/m, 31º50’S 117º37’E, Mount Male. Unknown. Stirling, 2.vi.1986; Le/Pe/m, P, Pm, 31º45’S 116º27’E, Bakers Female. Teneral abdomen, thorax and legs pale brown. Hill, 4.vii.1968, 6P, same loc., 24.vii.1969; Le/Pe/m, Le/Pe/f, Mensural features as in Table 2. 31º26’S 117º31’E, Yorkrakine Rock, 26.vii.1985, Le/Pe/m, same Pupa. Pale yellow-brown, apophyses weakly indicated, loc., 31.v.1986; Le/Pe/m, Pe/m, Le/Pe/m, Le/Pe/f, 30º25’S thoracic horn (Fig. 6G) golden brown. Abdominal spines and 116º58’E, Petrudor Rocks, 2.vii.1989, 2Pe, 4L(P), same loc., spinules golden brown. Frons, anteromedian thorax and prealar 11.vii.1989, Pe/m, 4L, same loc., 2.vii.1990. projection rugulose. Tergites as in B. grapeth (Fig. 3A) but with Etymology. Named for the rock-loving nature of this sparser conjunctival spines, sternites as in Fig. 7H. Mensural species, which is found exclusively in temporary streams features as in Table 3. flowing on rock outcrops. Larva. Head capsule pale yellow, with medium brown Male. Abdomen and thorax dark brown, with vittae no darker; mentum and apical 1/3 of mandible. Antenna yellow, with legs medium brown. Hypopygium (Figs 1E, 2E) with virga very Lauterborn organ half length of segment 3. Occipital margin dark small to apparently absent, anal point about 10 µm long, tergite brown. Premandible golden brown, with two apical teeth; IX with 6 or 7 moderately strong setae, and gonocoxite with mandible with four inner teeth and pale mola. Mentum (Fig. 8B) quite prominent bare volsella. Mensural features as in Table 1. with broad median tooth, with outermost tooth pointed and Female. As male, except in generic antennal and genitalic lying on line of even slope of mentum. Ventromental plate well features. Mensural features as in Table 2. developed, beard of 6-8 broad, median-lengthed branched setae Pupa. Pale yellow, apophyses golden-brown, thoracic horn (Fig. 9C). Anterior and posterior claws medium brown. Procercus (Fig. 6F) dark golden brown. Abdominal spines and spinules pale, apical setae golden. Mensural features as in Table 4. golden brown. Frons strongly rugose, anteromedian thorax and Comments. Botryocladius freemani is known in the adult prealar projection rugulose. Exceptionally three specimens have stage only by the teneral female, which may be distinguished by 6 well developed precorneal setae. Apex of anal lobe, bases of the apparent lack of bulbs prior to separately opening macrosetae, conventionally developed (Fig. 3H). Tergites as in spermathecal ducts, and perhaps also by the rather high value B. edwardsi (Fig. 5A) except for the weak development of for the anterior L.R., of 0.79 (this latter may however be armament on tergite II; sternites as in Fig. 7G. Mensural influenced by the pharate nature of the only measurable features as in Table 3. specimen. The pupa rather closely resembles that of B. grapeth Larva. Head capsule medium brown, with dark brown (q.v.) differing in the thoracic horn shape and in the lower mentum and apical 2/3 of mandible. Antenna brown, with density of spinules on the sternal conjunctives (compare Fig. Lauterborn organ slightly shorter than segment 3. Occipital 7H with Fig. 3F). The larva of B. freemani is the only species in margin brown, darker than head capsule. Premandible pale to the genus that has a simple, dome-shaped, median mental tooth. golden, with one apical tooth; mandible with 3 inner teeth and Although this initially caused it to be allocated to close to the darkened mola. Mentum with paired median teeth, often worn genus Acricotopus (Cranston, 1996) the ventromental beard to appear as single, with outermost tooth pointed and lying on setae are branched, as in larval congeners. Unreared eastern line of even slope of mentum. Ventromental plate small, with Australian larvae resembling B. freemani may belong to this beard of 6-8 branched setae of moderate length (Fig. 9D). taxon, but appear to have shorter ventromental setae. Rearing of Anterior and posterior claws golden brown. Procercus pale, this seemingly rare taxon will be required to allocate apical setae darker. Mensural features as in Table 4. appropriately. Comments. Botryocladius petrophilus is distinctive as a male by the very short to absent virga and the rather straight inner Botryocladius edwardsi sp.n. contour to the bare surfaced basal gonocoxite. The female may be distinguished by the larger number of strong setae on TIX Material examined. Holotype Pm, ARGENTINA, 4Pe, 16km (12-17) compared to six or less. The pupa lacks needle-like S. San Martin, Arroyo Partida, 21.i.1997, drift net (Cranston). spines, and can be distinguished amongst this grouping by the Paratypes, 5Pe, same data and slide as holotype. 10Pe, CHILE, spinose conjunctive to tergite II, shared with B. bibulmun, q.v. nr Peulla, Rio Canteras, 18.i.1986 (Edward); Pe, Prov. for separation based on the thoracic horn shape and sternite Llanquihue, lago Todos Los Santos, bei Puella, Mündung Rio armament. Bonito, CR53, 5.xii.1969 (Reiss) (FR); Pe, 72º56” 46º 43”S, Leones Valley, Rio Meliquina, 225 m.asl, (Brooks) (BMNH) Botryocladius freemani sp.n. Non-types: Pe, Chile: 72º56” 46º 43”S, Leones Valley, Rio Meliquina, 225 m.asl, (Brooks) (Santiago Museum) ?Acricotopus, Cranston, 1966. Etymology. This species is named for F.W. Edwards, who Material examined. Holotype Le/Pf, AUSTRALIA: pioneered the study of Patagonian Chironomidae based on a visit 33º41’07S 115º11.17’E, south-west Western Australia: Carbunup to the Bariloche region in 1926-7 R., 7.vii.1994 (Edward). Paratypes (all coll. Edward): 2Le/Pf, Adult male (teneral). Colour and features of wing as holotype (1 to ANIC); L, 34º54.20"S 117º58’02"E, Albany, unrecordable. Mensural features observable: antennal segments Bakers Junction Reserve, unnamed stream, 20.iv.1994; L, 1-12: 308 µm, 13: 99 µm: A.R. 0.32. Thorax with 2 scalpellate 32º32’S 116o02’E, Linwood Bk., 26.vi.1990; L, L(P), same loc., ac, 5dc, 4sc, 3pa. Hypopygium (Fig. 2F) with virga 27 µm long, 11.x.1991; L, 32º31’S 116º03’E, Cronin Bk.5.vii.1980; 4L, anal point 7 µm long, tergite IX with 6 or 7 moderately strong 32º30’S 116o04’E, Dillon Brook, 26.vi.1990 (ANIC); L, same setae, and gonocoxite with prominent rectangular volsella Botryocladius mapuche sp.n. completely overlying rounded ventral setose part of lobe. Pupa. Pale golden brown, apophyses pale brown, thoracic Material examined. Holotype Pe ARGENTINA, 41º01’S horn (Fig. 6H) darker brown. Abdominal spines and spinules 71º49’W, Puerto Blest, L. Nahuel Huapi, 2.i.1997 (Cranston) darker brown than cuticle. Frons, anteromedian thorax and prealar (Same slide as holotype of species IV). Paratypes, 2Pe, as projection all weakly rugulose. Tergites as in Fig. 5A, sternites holotype; CHILE, nr Peulla, 3Pe, Rio Canteras, 18.i.1986 as in Fig. 7I. Mensural features as in Table 3. (Edward); 3Pe, Lago Todos los Santos, Mündung Rio Bonito, Larva. Unknown 5.xii.1969 (Reiss) (FR); Pe, XI Region, Salto Velo de Novia, Comments. This is the only species of Neotropical 40km E. Pt Aisen, 13.i.1996 (Andersen) (ZMBN). Botryocladius in which the male is known, albeit from a pharate Etymology: Named for the indigenous people of the region, specimen. It is distinguished from Australian species by the the Mapuche; to be treated as a noun in apposition. A.R. (0.32), and by the rectangular shape of the inferior volsella, Adult. Unknown in contrast to the bulging lobe of extra-limital taxa. The shape Pupa. Medium brown thorax, paler abdomen, apophyses and length of the virga is also distinctive. brown, thoracic horn (Fig. 6J) with few tubercles, pigment The pupal exuviae can be distinguished amongst Neotropical variable from paler than thorax to filled with darker fluid. species by the armament of tergite II alone. The presence on Abdominal spines and spinules medium brown, darker than tergite II of a conjunctival spinule band, posterior transverse band cuticle. Frons, anteromedian thorax and prealar projection smooth of broad, not needle-like, spines (< 25 µm long), and widely to very weakly rugulose. Tergites as in Fig. 5C, sternites as in distributed anteromedian spinule shagreen separates B. edwardsi Fig. 7K. Mensural features as in Table 3 not only from Neotropical species, but from two otherwise Larva. Unknown somewhat similar Australian species. The group of spines on Comments. This taxon is known only from the pupal the posterior of sternite VIII of male exuviae (Fig. 7I) is found exuviae, which can be distinguished by the development of the otherwise only in B. grapeth and B. glacialis. posterior tergal spine band as needle-like, fine spines > 50 µm long, and the lack of conjunctival armament on tergite II (which Botryocladius glacialis sp.n. differentiates this species from Australian taxa with needle-like spines). Botryocladius mapuche is distinguished from the Material examined. Holotype Pe ARGENTINA, 41º02’S otherwise very similar B. tronador by the thoracic horn bearing 71º49’W, nr. Bariloche, Lago Frias, 3.i.1997 (Cranston). few tubercles restricted to the apical part (Fig. 6J, compare Fig. Paratypes, Pe, same data and slide as holotype; 3Pe, Rio Negro, 6K), the more densely armed tergite IV with the typical 5 D nr Bariloche, Cascado los Alerces, 31.xii.1996 (Cranston & setae in B. mapuche (Fig. 5C, compare Fig. 3E), and by some Edward); 16Pe, Rio Caliefu, Casa de Pedra, 21.i.1997 minor differences in the sternal armament (Fig. 7K, compare (Cranston). CHILE, 5Pe, Los Lagos, Prov. Llanquihue, Lago Fig. 7L). Todos los Santos, bei Peulla, Mündung Rio Bonito, CR 53, 5.xii.1969 (Reiss) (FR); 2Pe, Lago Riñihue, am Ausfluss, Botryocladius tronador sp.n. 5.xi.1969 (Reiss) (FR). Etymology. This species is named for its known locations: Material examined. Holotype Pe ARGENTINA, 41º01’S Andean glacial-fed lakes and streams. 71º49’W, Puerto Blest, L. Nahuel Huapi, 2.i.1997 (Cranston). Adult. Unknown. Paratypes, 11Pe, as holotype; 4Pe, ARGENTINA: Rio Caleufu, Pupa. Pale brown, apophyses dark brown, thoracic horn (Fig. Casa de Piedra, 2.i.1997(Cranston). 6I) no darker than thoracic cuticle. Abdominal spines and spinules Etymology. Named for the dominant glaciated mountain at golden brown. Frons, anteromedian thorax and prealar this latitude in the Andes, where it overlooks virtually all of the projection rugulose. Sternite VIII of male posteriorly with collection sites for the Neotropical specimens described here. cluster of spines, female without. Tergites as in Fig. 5B, sternites Adult. Unknown as in Fig. 7J. Mensural features as in Table 3. Pupa. Medium brown thorax and abdomen, apophyses Larva. Unknown brown, thoracic horn (Fig. 6K) weaker pigmented than thorax. Comments. This taxon is known only from the pupal Abdominal spines and spinules brown, darker than cuticle. Frons, exuviae, which can be distinguished amongst Neotropical anteromedian thorax and prealar projection weakly rugulose. species by the weak armament of tergite II and the broad, not Tergites as in Fig. 5C, excepting tergite IV (Fig. 3E), sternites as needle-like, posterior spines of tergites III-VIII. Other taxa in Fig. 7L. Mensural features as in Table 3 sharing these features are Australian, and may be distinguished Larva. Unknown by the weaker or undeveloped spines on sternite III, and notably Comments. This taxon is known only from one pupal by the weaker posterior spines on tergite VIII. The length and exuviae, which can be distinguished amongst Neotropical semi-taeniate development of the thoracic setae, notably the species by the needle-like spines on the posterior tergites frontals, is unique in B. glacialis. III-VIII. It resembles B. mapuche except in the increased number The sternal armament of B. glacialis (Fig. 7J) including of D setae and pattern of armament on tergite IV (Fig. 3E) and strong bands of spines on sternites III-VII and well developed in the stronger development of sternal shagreen (Fig. 7L). conjunctival bands on IV-VI is stronger than in any other Neotropical species. The unusual development of the anal lobe Patagonian larval species I apex (Fig. 3G) seen in B. bibulmun is also found in B. glacialis. There is variation in the development of the conjunctival Material examined. ARGENTINA, 1Le, 16km S. San spinule band on tergite II, ranging from apparently absent to Martin, Arroyo Partida, 21.i.1997, drift net (Cranston). moderately strongly uni- to biserially developed. A unique Larva. Mandible as in Fig. 8H. Mentum as in Fig. 9F. development in one of the female specimens from Lago Frias is Mensural features as in Table 4. the absence of a tergal conjunctive spine row on tergite VI. Sexual Comments. This specimen, a larval exuviae taken by drift dimorphism is evident, with the posterior spine patch on sternite netting, represents the only putative larva of this grouping from VIII developed only in the male. South America. It somewhat resembles the generic diagnosis derived from Australian species, except for the mandible (Fig. 8H) and ventromental area (Fig. 9F). The mandible has an RI of 0.50, in which the consensus reduced Botryocladius / apical tooth lying on a different plane and shorter than the first “SO2”/ (“SO3” (“MO5” ( Synorthocladius + Paralimnophyes))) of three inner mandibular teeth. The exuvial mentum is split to the unresolved relationship portrayed in Fig. 11. medially and it is impossible to reconstruct the median part. The In species-level analysis, using characters/states (Appendix ventromental beard is strong, with some 8 setae, more branched 2) represented in matrix Table 6 and with “SO3” as outgroup, than in any other species (Fig. 9F) and with the postero-lateral revealed six trees with length 55 steps, CI of 0.52 and RI of mentum, outermost mental tooth, and ventromental plate more 0.71 with a consensus cladogram shown in Fig. 12. Under complex than in Australian Botryocladius species (Fig. 9A-E). successive approximation weighting, the two resultant trees This ventromental plate starts to approach the complexity seen differed from each other and from the consensus tree only with in the Australian taxon “SO2” (Cranston, 1996) and this larva respect to arrangements of bibulman / petrophilus / edwardsi). may be associated with an undescribed taxon, possibly No clades were significant under T-PTP testing. congeneric with “SO2”, known as pupa and pharate adult from several Patagonian lotic sites. Discussion

Ecology and distribution In analysis 1 our interest lay in Botryocladius and since our matrix was designed to elucidate its immediate relationships we The immature stages of Botryocladius have been found in a make no claims for thorough study of other taxa. However, it is variety of habitats, ranging from glacial Neotropical lakes and of some interest that Paralimnophyes is suggested to lie outside streams, to slow-flowing waters in Australia. Identification of the Limnophyes + Compterosmittia group in keeping with lentic habitats by the recovery of pupal exuviae from lakes has Rossaro (1990) who also found Paralimnophyes did not cluster been confirmed by larval collections only in one Australian lake. with Limnophyes. These hypothesised placements contrast to Since Neotropical lake sites were all close to outlets of fast the Heleniella (Limnophyes (Paralimnophyes + flowing glacial torrents, exuviae might have derived from Compterosmittia)) arrangement that Sæther (1990b) proposed running waters but certainly there must be truly lentic species of in an analysis limited at generic level to these four genera and Botryocladius. with information lacking on certain immature stages. However In Australia, where the larval biology in lotic systems is well as assessed by T-PTP testing, no groupings were significant, known for several species, the larvae can be quite abundant in and the results must be considered tentative. temporary (seasonal) and permanent lotic waters, predominantly Taking the results from analysis 1 (Fig. 11) as indicating in the cooler and forested parts of the country (the south-east that “SO2” and “SO3” are appropriate putative outgroups, the and south-west). The exception to this apparent preference for second analysis concerned the structure of the species-level shaded cooler waters is B. petrophilus, which lives in water films terminal taxa. An unexpected position of taxon “SO2” deeply of streams flowing over rockfaces that may be exposed to strong embedded within Botryocladius clearly is an artefact of the sun (e.g. Kata Tjuta in central Australia). However, in Western restriction of the data set to pupal features, and particularly to Australia B. petrophilus is present only in winter/spring, when the polarity assigned by outgroup “SO3” to the many “absences” temperatures are lower and seasonal rainfall encourages stream of sternal characters for “SO2”. This was confirmed in an analysis flow. (not shown) in which polarity estimated from an all-zero outgroup There is some evidence for the eastern Australian B. grapeth, (reflecting character state distribution within a broader B. mdfrc and B. brindabella having some association with grouping) resulted in postulation of “SO2” as sister to immersed wood, and the gut contents of B. grapeth frequently Botryocladius. Morphological features of “SO2” that refute the contain wood fibres and fragments of shredded leaves (McKie, pupal embedded position include the quite different larval 1996, P. S. Cranston, personal observation). This is not so for ventromental plate construction which has a typically the Western Australian species, whose larval guts contain Limnophyes-like lobe lying between the narrow ventromental amorphous materials (D.H.D. Edward, personal observation. plate proper and the outer mental teeth, and the possession of a Botryocladius bibulmun has two emergence periods in the year simple labral S1. In the adult the strength of the anal point in the jarrah forest streams. differs from that of all known Botryocladius males, and a In Western Australia no two Botryocladius species co-exist further difference is the apparent lack of any acrostichal setae in the same water body, but the eastern widespread species B. (difficult to see in teneral specimen available). These features, grapeth co-occurs with most other species, including, at one site, in conjunction with the non-botryose pupal thoracic horn, the temporary stream taxon B. petrophilus. The isolated appear to render the inclusion of “SO2” within Botryocladius occurrence of B. grapeth in Western Australia is superficially spurious. surprising, but coincides with several relictual taxa in the Examination of the structure of the pupal cladogram shows Stirling Range streams, including the only Western Australian that Australian and Patagonian taxa are interspersed in several record of the widespread eastern species Eukiefferiella insolita clades, thus B. tasmania (Tasmania) and B. glacialis (Patagonia) Freeman (Boothroyd & Cranston, 1995). are putative sister species, Patagonian B. edwardsi lies with B. petrophilus (central Australian) and B. bibulman (s.w. W.A.). Cladistic analysis The Patagonian sister taxa B. tronador and B. mapuche form the sister to a larger clade including south American-Australian taxa The generic-level analysis, using characters/states whose resolution depends upon treating multistate characters as (Appendix 1) represented in matrix Table 5 and with Diplocladius additive. Botryocladius is unusual amongst the analysed as outgroup and all characters additive, revealed trees of length Gondwanan clades present in south-west Western Australia in 107 steps, CI of 0.44 and RI of 0.48 in all cases with having closer sister-group relationships to Patagonian taxa than Botryocladius sister to “SO2”, with “SO3” the sister at next to south-east Australian taxa (Edward, 1989). remove. This clade is sister (amongst the taxa selected for The possibility that trans-Antarctic clades are artefactual has inclusion) to ”MO5” ( Synorthocladius + Paralimnophyes). been tested by assessing the number of additional Support for these clades was non-significant under T-PTP synapomorphies that must be added to the matrix to produce testing. Treatment of multistate characters 12, 15, 16, 17 & 39 Australian or Patagonian geographic monophyly. The result of as non-additive gave trees of length 103 steps, CI of 0.46 and 2-6 extra steps (according to whether multistate characters are non-additive (2) or additive (6)) suggest that this is very The senior author is grateful to the Australian Academy of unlikely. Thus the genus can be postulated to have originated in Sciences for support to visit the Natural History Museum, south-eastern Gondwana with some species formation prior to London, and the Zoologische Staattssamlung, Munich, to breakup. This gave rise to several trans-Antarctic distributed examine their collections. The Australian Biological Resources ancestors that subsequently vicariated into sister taxa on each Study (ABRS) funded illustrations of some Australian species. side of the fragmented continents. Apparent absence from New Thanks are due to Wendy Lee, who verified the distributions Zealand (I. Boothroyd, personal communication) suggests that and databased all specimens in the NAIC database. Aimorn unless there was ancient presence and subsequent extinction Stewart prepared the scanning electron microscope image. Robert (deemed unlikely given Brundin’s (1966) elucidation of retained Hoare kindly provided advice on naming of taxa, but no blame presence of many ecologically similar cold stenotherm should be ascribed to him. podonomines and diamesines), the genus originated after New Zealand’s isolation from Austro-Antarctic and prior to References Australian isolation from S. America-Antarctica. The geological scenario (derived from Coleman, 1980; Stevens, 1989; Boothroyd, I.K.G. & Cranston, P.S. (1995) Two Veevers. 1991; Walley & Ross, 1991) has Australia separating Orthocladiinae (Chironomidae) genera common to throughout the Cretaceous from the Antarctic through a New Zealand and Australia: Pirara n. gen. and west-to-east opening of the future Southern Ocean. The Eukiefferiella Thienemann. Chironomids: from Genes to Antarctic itself may have been divided by a Cretaceous seaway Ecosystems (ed. by P.S. Cranston), pp. 389-408. (Zinsmeister, 1987) and the geological evidence concerning CSIRO, Melbourne, mobility of faunas between South America and Australia through Brundin, L. (1966) Transantarctic relationships and their Antarctica and the effectiveness of barriers within the area is significance, as evidenced by chironomid midges with a unclear. Australia itself was covered with shallow seas, although monograph of the subfamilies Podonominae and the eastern cordillera was rising to give elevation and commence Aphroteniinae and the austral Heptagyiae. Kunglica the asymmetrical (dominantly westward) drainage pattern still Svenska Vetenskapsakademiens Handlingar, 11, 1-472 + seen today. The precise timing of the loss of contiguous, 30 plates. above-sea, connections between Australia and New Zealand (via Coffman, W.P., Cranston, P.S., Oliver, D.R. & Sæther, O.A. Tasmania, or the Lord Howe Rise) are unclear because of (1986) Keys and diagnoses of the pupae of the subfamily variations in marine regressions in the late Cretaceous (100-65 Orthocladiinae (Diptera, Chironomidae). Entomologica mybp) but a dating of about 80 mybp generally is agreed for Scandinavica Supplement, 28, 147-296. complete continental separation and new sea floor creation. After Coleman, P.J. (1980) Plate tectonics background to loss of connection to New Zealand, Australia remained in biogeographic development in the southwest Pacific over contact with South America through Antarctica until about 38 the last 100 million years. Palaeography, mybp, when the seaway connecting the Pacific and Indian Oceans Palaeoclimatology, Palaeoecology, 31, 105-21. was finally formed with the breaching of the South Tasman Rise, Cranston, P. S. (1994) Morphology, Chapter 2. In the last remnant connection between south-eastern Australia and ‘Chironomidae: Biology and Ecology of Non-biting Antarctic (Zinsmeister, 1987). Midges’ (eds P. D. Armitage, P. S. Cranston and L. C. V. The cool stenothermic conditions currently favoured by most Pinder.), pp. 11-30. Chapman and Hall, London, Glasgow, Botryocladius species evidently were present in the past, even New York, Tokyo, Melbourne, Madras. as the continental fragments moved northwards, due to (i) the Cranston, P. S. (1996) Identification Guide to the cooling of Antarctica, (ii) the development of the cool Chironomidae of New South Wales. AWT Identification Circum-Antarctic current and (iii) orogeny in southern South Guide Number 1. viii + 376 pp. Australian Water America and eastern Australia. Technologies Pty Ltd, Sydney. Thus it seems parsimonious that the current-day Cranston, P.S. & Edward, D.H.D. (1992) A systematic distribution of the genus Botryocladius derives from an reappraisal of the Australian Aphroteniinae ancestor distributed in eastern Gondwana at no earlier date than (Chironomidae) with dating from vicariance biogeography. 80 mybp. By 38 mybp the already-speciose clade was subjected Systematic Entomology, 17, 41-54. to vicariance by the fragmentation of the land mass and the Cranston, P.S. & Edward, D.H.D. (1998) Afrochlus Freeman - developing inhospitality of Antarctica. Such dating (and earlier) an African gondwanan midge, and the phylogeny of the scenarios have been drawn for other clades of Chironomidae Podonominae (Diptera; Chironomidae). Systematic notably by Brundin (1966), Cranston, Edward & Colless (1987) Entomology, 23, 77-90. and Cranston & Edward (1992). These concerned undoubted Cranston, P.S. & Kitching, R. (1995) The Chironomidae of basal clades in few subfamilies (Podonominae, Aphroteniinae), Austro-Oriental phytotelmata (plant-held waters): Richea whereas in contrast, Botryocladius is relatively derived within pandaniformis Hook.f.. Chironomids: from Genes to the Orthocladiinae, postulated as one of the most derived of Ecosystems (ed. by P.S. Cranston), pp. 225-32. CSIRO, subfamilies. Melbourne, Cranston, P.S., Edward, D.H.D. & Colless, D.H. (1987) Acknowledgements Archaeochlus Brundin: a midge out of time (Diptera: Chironomidae). Systematic Entomology, 12, 313-34. We are grateful to all agencies for the various permits Cranston, P.S., Oliver, D.R. & Sæther, O.A. (1989) Keys and required to collect these insects in national parks and state diagnoses of the adult males of the subfamily forests. Many specimens came from particular surveys, notably Orthocladiinae (Diptera, Chironomidae). Entomologica for the Australian Federal Government programme concerning Scandinavica Supplement 34, 165-352. monitoring of river health. Martin Spies and Freider Reiss kindly Edwards, F.W. (1931) Part II. Fascicle 5. Chironomidae. sorted and loaned material from the latter’s private collection Diptera of Patagonia and South Chile. pp. 232-331. for incorporation in this study and critically reviewed drafts of Edward, D.H.D. (1989) Gondwanaland elements in the this manuscript. We thank Ole Sæther and Ian Boothroyd for Chironomidae (Diptera) of south-western Australia. Acta providing useful comments and advice. Biologica Debrecina, Oecologica Hungarica, 3, 15,181-7. Faith, D.P. (1991) Cladistic permutation tests for monophyly and non-monophyly. Systematic Zoology, 40, 366-75. Faith, D.P. & Cranston, P.S. (1991) Could a cladogram this short have arisen by chance alone?: on permutation tests for cladistic structure. Cladistics, 7, 1-28. Farris, J.S. (1988) Hennig86 reference. Version 1.5. Port Jefferson, New York. Freeman, P. (1961) The Chironomidae (Diptera) of Australia. Australian Journal of Zoology, 9, 611-737. Langton, P. H. (1994) If not “filaments”, then what ? Chironomus 6, 9. Langton, P. H. & Cranston, P. S. (1991) Pupae in nomencla ture and identification: West Palaearctic Orthocladius s.str. (Diptera: Chironomidae) revised. Systematic Entomology 16, 239-52. Ramos, T.C. (1996) Tree Gardener 1.0. Privately distributed by Tiago Courrol Ramos, Museu de Zoologia, Universidade de Sao Paulo, Sao Paulo, Brasil. Rossaro, B. (1990) A numerical taxonomy study of Orthocladiinae (Diptera: Chironomidae). Acta Biologica Debrecen, Oecologica Hungarica, 2, 315-24. Sæther, O. A. (1977) Female genitalia in Chironomidae and other Nematocera: morphology, phylogenies, keys. Bulletin of the Fisheries Research Board of Canada, 197, 1-211. Sæther, O. A. (1980) A glossary of chironomid morphology terminology (Diptera: Chironomidae). Entomologica Scandinavica Supplement, 14, 1-51. Sæther, O. A. (1990a) Phylogenetic trends and their evaluation in chironomids with special reference to orthoclads. Acta Biologica Debrecen, Oecologica Hungarica, 2, 53-75. Sæther, O.A. (1990b) A review of the genus Limnophyes Eaton from the Holarctic and Afrotropical regions. Entomologica Scandinavica Supplement, 35, 1-135. Stevens, G.R. (1989) The nature and timing of biotic links between New Zealand and Antarctic in Mesozoic and early Cretaceous times. Origins and evolution of the Antarctic biota.(ed. by J.A. Crane) pp.141-66. Geological Society Special Publication 47. Veevers, J.J. (1991) Phanerozoic Australia in the changing configuration of Proto-Pangea through Gondwanaland and Pangea to the present dispersed continents. Australian Systematic Botany 4, 1-11. Walley, A.M. and Ross, M.I. (1991) Preliminary reconstructions for the Cretaceous to Cainozoic of the New Zealand - New Caledonia region. Palaeogeography, 31, 1-43. Zinsmeister, W.J. (1987) Cretaceous palaeogeography of Antarctica. Palaeography, Palaeoclimatology, Palaeoecology, 59, 197-205. Fig 1. Botryocladius petrophilus. A. wing. B. cibarial pump and tentorium. C. thorax. D. posterior tibial apex. E. genitalia, left side = dorsal view, right side = stylized ventral view. Fig. 2. Botryocladius. Adult genitalia. A-E, male hypopygia, contour of inner margin of gonocoxite and virga; dorsal view. A. B. grapeth, B, B. brindabella, C. B. bibulmun; D. B. collessi, E. B. petrophilus, F. B. edwardsi; G-H, female genitalia, B. grapeth, G. ventral view, H. detail. Fig. 3. Botryocladius pupae. A. B. grapeth, dorsal abdomen, B. B. tasmania, cephalic area, C. B. mdfrc “mace” on tergite IV, D. B. australoalpina, left side of tergites II and III, E. B. tronador left side of tergites III and IV, F. B. grapeth sternites, G-H apex of anal lobe and bases of macrosetae of G. B. bibulmun, H. B. petrophilus. Fig. 4. Botryocladius, pupal dorsal abdomen. A. B. brindabella, B. B. collessi, C. B. mdfrc. Fig. 5. Botryocladius, pupal dorsal abdomen. A. B. edwardsi, B. B. glacialis, C. B. mapuche. Fig. 6. Botryocladius, pupal thoracic horns. A. B. grapeth, B. B. collessi, C. B. tasmania, D. B. australoalpina, E. B. bibulmun, F. B. petrophilus, G. B. freemani, H. B. edwardsi, I. B. glacialis, J. B. mapuche, K. B. tronador. Fig. 7. Botryocladius, pupal ventral abdomen. A. B. brindabella, B. B. mdfrc, C. B. collessi, D. B. tasmania, E. B. australoalpina, F. B. bibulmun, G. B. petrophilus, H. B. freemani, I. B. edwardsi, J. B. glacialis, K. B. mapuche, L. B. tronador. Fig. 8. Botryocladius larvae. A. B. grapeth antenna, B-C. B. freemani, B. mentum, C. mandible. D-E. B. grapeth, D. mentum, E. mandible. F-G. B. brindabella, F. mentum, G. mandible. H. Patagonian larval sp., mandible, I. B. freemani, labrum and epipharynx, J. B. freemani, maxilla. Fig. 9. Botryocladius larval menta. A. B. grapeth, B. B. bibulman, C. B. freemani, D. B. petrophilus, E. B. brindabella, F. Patagonian larval sp. Fig. 10. Botryocladius freemani. Sensory setae of labrum and epipharynx (scanning electron microscopy). Fig.11. Postulated phylogeny of Botryocladius and putative generic relatives, based on data matrix 1, characters in Appendix 1. Codes refer to taxa in Cranston (1996). Fig. 12. Postulated phylogeny of Botryocladius species based on data matrix 2, characters in Appendix 2. Codes refer to taxa in Cranston (1996). Characters used in phylogenetic analyses. Appendix 1 Adult 1. Palp sensilla chaetica: (0) absent; (1) present. 2. Antepronotum: (0) weak; (1) well developed. 3. Antepronotal lobes: (0) separated; (1) joined. 4. Dorsal antepronotals: (0) present; (1) absent. 5. Lateral antepronotals: (0) absent; (1) present. 6. Acrostichals: (0) present; (1) absent. 7. Acrostichals: (0) simple; (1) scalpellate. 8. Wing punctation: (0) fine; (1) coarse. 9. R4+5 ending relative to M4+5: (0) distal; (1) above/proximal. 10. Cu1: (0) straight; (1) curved. 11. Squama: (0) fully fringed; (1) reduced. 12. Costal extension: (0) absent; (1) weak; (2) strong. 13. Pseudospurs: (0) absent; (1) present. 14. Sensilla chaetica (legs): (0) absent; (1) present. 15. Pulvilli: (0) absent; (1) weak; (2) strong. 16. Anal point: (0) absent; (1) weak; (2) strong. 17. Virga: (0) absent; (1) weak; (2) strong. 18. Seminal capsules: (0) bare; (1) microtrichiose. 19. Seminal capsules: (0) with bulb; (1) without bulb. 20. Seminal ducts: (0) without loop; (1) with oral loop. Pupa 21. Frontal setae arise: (0) direct; (1) from tubercles; (2) absent. 22. Thoracic horn: (0) present; (1) absent. 23. Tergite I: (0) bare; (1) spinulose. 24. Sternites: (0) bare; (1) spinose; (2) fine shagreen. 25. Tergal conjunctives: (0) bare; (1) only III spinose; (2) most spinose. 26. Pedes spurii A (0) absent; (1) present. 27. Pedes spurii B: (0) absent; (1) present. 28. Anal lobe: (0) fringed; (1) 3 macrosetae. 29. Needle-like tergal spines: (0) absent; (1) present. 30. Tubercular tergal armament: (0) absent; (1) present. 31. Posterior hooklets on TII: (0) present; (1) absent. Larva 32. 6th antennal segment: (0) absent; (1) present. 33. Antennal segment 3 : 4 (0) longer/subequal; (1) shorter. 34. Lauterborn organs: (0) distinct; (1) weak/absent. 35. SI seta: (0) simple; (1) bifid; (2) multiple branched. 36. Premandibular teeth: (0) 1-2 ; (1) > 2. 37. Premandibular brush: (0) present; (1) absent. 38. Ventromental beard: (0) present; (1) absent. 39. Ventromental plates: (0) distinct; (1) weak; (2) absent. Appendix 2 1. Tergal spines: (0) needle-like; (1) broad. 2. Conjunctive II: (0) bare; (1) spinose. 3. Conjunctive II spines: (0) transverse; (1) divided medially (? = n.a.). 4. Conjunctive IV spines: (0) transverse; (1) broadened medially. 5. T III L setae:{0) thin,; (1) semi-taeniate. 6. Sternite II: (0) bare; (1) spinulose. 7. Sternite III: conjunctive (0) bare; (1) spinose. 8. Sternite III: (0) bare; (1) spinulose; (2) spine row. 9. Sternite IV: (0) bare; (1) spinulose; (2) with spine row. 10. Sternite V: (0) bare; (1) spinulose; (2) with spine row. 11. Sternite VI: (0) bare; (1) spinulose; (2) with spine row. 12. Sternite VII: (0) bare; (1) spinulose; (2) with spine row. 13. Sternite VIII: (0) bare; (1) spinulose; (2) with spine row. 14. Anal macrosetae: (0) longer than anal lobe; (1) shorter than anal lobe. 15. Frontal setae: (0) present; (1) absent. 16. Precorneal setae: (0) subequal; (1) one (pc3) shortened. 17. Anal macrosetal bases: (0) normal; (1) crenulate. 18. Thoracic horn shape: (0) globular; (1) elongate; (2) triangular. 19. Thoracic horn neck: (0) narrow; (1) broad. 20. Thoracic horn: (0) pale; (1) dark. 21. Thoracic horn: (0) smooth; (1) weakly tuberculose; (2) strongly tuberculose. Table 1. Mensural features of Botryocladius males. Abbreviations as in text. grapeth brindabella collessi bibulman petrophile n = 4-6 1 1 5 6 B.l 2.3-2.8 2.10 3.70 1.9-2.5 2.4-2-8 W.l 1.2-1.5 1.30 ? 1.1-1.4 1.4-2.1 Fl.13 200-332 276 440 248-390 350-600 Fl.1-12 437-505 395 440 410-450 415-480 A.R. .46-.70 0.70 1 .59-.87 .83-1.25 Clyp 8-10 7 14 9-13 9-16 P-orb 4-6 6-7 7 3-4 5-7 Palp 1 25-30 25 ? 24-32 24-30 Palp 2 23-40 40 48 20-44 44-56 Palp 3 50-78 78 73 52-70 74-96 Palp 4 78-100 86 100 74-90 72-88 Palp 5 108-135 140 140 118-140 100-120 L.aps. 3-7 2 3 1 1-2 Ac 2 3 2 1-2 3-8 Dc 6-9 6 13 5-8 7-8 Pa33 533-5 Scts 5-9 4 9 4-5 4-8 Cu1 520-730 625 ? 495-655 680-832 M 410-520 465 ? 400-545 560-688 V.R. 1.24-1.40 1.34 ? 1.19-1.28 1.15-1.25 R 4-7 6 ? 1-5 2-8 Sq 3-6 1-3 ? 2-4 4-8 Gcx 140-150 156 225 120-150 196-248 Gst 65-67 74 95 54-64 86-116 Virga 15-25 8 15 14-24 0-5 P1 Fe 430-500 465 ? 430-545 520-760 Ti 550-620 550 608 480-615 585-890 Ta1 320-400 390 440 335-410 390-590 Ta2 220-280 250 ? 225-255 230-375 Ta3 160-200 185 ? 175-215 190-280 Ta4 100-130 110 ? 70-110 120-185 Ta5 60-90 70 ? 65-80 90-110 LR 0.64-0.69 0.63 0.72 0.63-0.70 0.59-0.67 P2 Fe 430-500 470 ? 390-510 535-750 Ti 420-480 480 560 400-520 550-785 Ta1 220-280 250 288 200-280 255-360 Ta2 120-160 145 ? 105-160 160-230 Ta3 90-120 105 ? 90-120 95-185 Ta4 60-70 70 ? 56-80 90-135 Ta5 50-70 65 ? 55-70 90-105 LR 0.51-0.58 0.52 0.60 0.50-0.54 0.43-046 P3 Fe 440-480 520 ? 415-585 575-830 Ti 50-620 560 640 490-650 615-910 Ta12 80-350 310 384 270-350 345-495 Ta2 160-200 185 ? 150-200 210-305 Ta3 130-170 150 ? 135-185 175-240 Ta4 70-100 90 ? 65-105 95-150 Ta5 70-80 70 ? 55-90 80-110 LR 0.54-0.56 0.56 0.60 0.54-0.59 0.50-0.56 P1 spur 32-42 36 52 30-40 42-62 P2 spur 15-20 16 22 12-20 18-22 P2 spur 13-18 16 22 12-20 14-20 P3 spur 40-48 40 50 36-40 40-48 P3 spur 13-20 10 16 12-18 16-20 Table 2. Mensural features of Botryocladius females. Abbreviations as in text. grapeth brindabella collessi bibulman petrophile n =4-61156 B.l 2.3-2.8 2.10 3.70 1.9-2.5 2.4-2-8 W.l 1. 2-1.5 1.30 ? 1.1-1.4 1.4-2.1 Fl. 13 200-332 276 440 248-390 350-600 Fl 1-12 437-505 395 440 410-450 415-480 A.R. 0.46-0.70 0.70 1 0.59-0.87 0.83-1.25 Clyp 8-10 7 14 9-13 9-16 P-orb 4-6 6-7 7 3-4 5-7 Palp 1 25-30 25 ? 24-32 24-30 Palp 2 23-40 40 48 20-44 44-56 Palp 3 50-78 78 73 52-70 74-96 Palp 4 78-100 86 100 74-90 72-88 Palp 5 108-135 140 140 118-140 100-120 L.aps. 3-7 2 3 1 1-2 Ac 2 3 2 1-2 3-8 Dc 6-9 6 13 5-8 7-8 Pa 3 3 5 3 3-5 Scts 5-9 4 9 4-5 4-8 Cu1 520-730 625 ? 495-655 680-832 M 410-520 465 ? 400-545 560-688 V.R. 1.24-1.40 1.34 ? 1.19-1.28 1.15-1.25 R 4-7 6 ? 1-5 2-8 Sq 3-6 1-3 ? 2-4 4-8 Gcx 140-150 156 225 120-150 196-248 Gst 65-67 74 95 54-64 86-116 Virga 15-25 8 15 14-24 0-5 P1 Fe 430-500 465 ? 430-545 520-760 Ti 550-620 550 608 480-615 585-890 Ta1 320-400 390 440 335-410 390-590 Ta2 220-280 250 ? 225-255 230-375 Ta3 160-200 185 ? 175-215 190-280 Ta4 100-130 110 ? 70-110 120-185 Ta5 60-90 70 ? 65-80 90-110 LR 0.64-0.69 0.63 0.72 0.63-0.70 0.59-0.67 P2 Fe 430-500 470 ? 390-510 535-750 Ti 420-480 480 560 400-520 550-785 Ta1 220-280 250 288 200-280 255-360 Ta2 120-160 145 ? 105-160 160-230 Ta3 90-120 105 ? 90-120 95-185 Ta4 60-70 70 ? 56-80 90-135 Ta5 50-70 65 ? 55-70 90-105 LR 0.51-0.58 0.52 0.60 0.50-0.54 0.43-046 P3 Fe 440-480 520 ? 415-585 575-830 Ti 50-620 560 640 490-650 615-910 Ta1 280-350 310 384 270-350 345-495 Ta2 160-200 185 ? 150-200 210-305 Ta3 130-170 150 ? 135-185 175-240 Ta4 70-100 90 ? 65-105 95-150 Ta5 70-80 70 ? 55-90 80-110 LR 0.54-0.56 0.56 0.60 0.54-0.59 0.50-0.56 P1 spur 32-42 36 52 30-40 42-62 P2 spur 15-20 16 22 12-20 18-22 P2 spur 13-18 16 22 12-20 14-20 P3 spur 40-48 40 50 36-40 40-48 P3 spur 13-20 10 16 12-18 16-20 Table 3. Mensural features of Botryocladius pupae. Abbreviations as in text. grapeth brindabella mdfrc collessi tasmania a-alpina bibulman petrophile freemani edwardsi glacialis mapuche tronador n =1237598141338481 b.l. 2.3-3.4 2.2-3.4 2.6-3.4 3.3-4.2 3.0-4.0 3.4-3.9 2.1-2.9 2.9-4.0 2.4-3.6 2.4-2.8 3.6-4.1 2.1-2.8 2.7 fs.l 50-70 65-90 75-95 95-105 45-85 abs 35-54 45-115 85-90 90-105 210-245 60-75 95 th.h.l. 75-125 125-155 120-165 150-215 135-165 85-110 60-105 120-205 95-115 125-150 125-180 125-130 115 apn1 65-85 90-155 135-155 140-164 75-100 40-70 40-95 85-165 125 100-115 205-270 65-90 100-110 apn2 70-95 100-150 90-155 130-165 65-95 50-75 40-90 80-130 95 85-115 190-260 90-105 - apn3 45-70 60-80 45-70 75-95 5 0-85 abs 30-50 50-85 abs 70-95 90-155 35-70 50-60 pc1 65-100 120-150 125-155 140-170 85-110 60-105 60-85 80-165 140 100-135 210-280 55-95 110-125 pc2 40-65 75-130 50-100 85-105 60-80 35-70 35-70 35-110 ? 75-105 90-140 55-65 55 pc3 70-95 105-120 100-165 120-170 75-105 25-35 35-71 70-145 ? 105-125 205-250 70-95 105-115 dc1 30-45 50 40-55 40-55 35-40 30-45 20-30 30-70 40 30-45 45-105 30-35 35 dc2 30-65 60-75 50-70 65-85 60-85 50-60 20-40 50-95 55 65-95 85-140 50-70 45 dc3 20-30 50-60 40-50 35-45 25-35 30-35 15-35 45-65 30 30-40 45-70 25-30 - dc4 20-35 35-60 35-40 35-40 20-35 30-40 10-30 35-70 30 40-50 25-45 25-30 35 dc1-2 10-30 20-40 15-35 20-55 30-35 30-35 15-30 15-75 8-19 25-50 35-60 15-20 20 dc2-3 90-135 75-145 105-130 110-185 125-175 100-115 75-115 90-175 80-90 80-120 140-190 110-135 115 dc3-4 10-15 10-25 10-20 10-50 5-20 10-15 10-20 5-55 5-10 10-35 10-30 10-25 - ms.l 160-210 200-240 215-265 220-290 195-235 110-120 170-210 225-365 190-215 180-200 235-330 115-145 185 Table 4. Mensural features of Botryocladius larvae. Abbreviations as in text.

grapeth brindabella bibulman petrophile freemani n = 8 1 10 16 12 B.l. 3.4-5.3 ? 3.0-3.8 4.0-5.8 3.1-4.8 H.l. 330-380 350 295-315 360-490 310-375 Ant 1 29-35 50 30-34 33-47 38-46 Ant 2 10-13 12 11-14 10-14 8-11 Ant 3 4-8 6 3-5 3-5 4-6 Ant 4 5-8 8 6-10 6-8 8-10 Ant 5 4-6 6 5-6 3-6 5-6 Ant 6 0-2 absent 5-10 absent absent A.R. 0.84-1.3 1.56 0.72-0.96 1.4-1.7 1.47-1.54 Bl 22-27 35 27-30 21-25 22-27 RO 5-8 5-6 4-8 10-11 5-7 Md 108-128 140 110-117 112-134 110-115 M.w. 88-106 105 88-99 88-115 82-93 M.m.w.20-26 21 21-25 19-24 26-31 vms.l 40 10 35 36 21 An l. 290-355 ? 290-385 230-380 245-300 Pc.l 11-14 ? 10-12 14-16 10-14 Pc.w 10-14 ? 11-14 14-18 10-14

Table 5. Character states of selected Orthocladiinae genera. See Appendix 1 for character states.

5 10 15 20 25 30 35 Diplocladius ?001111?0011000100110000111100000020100 Botryocladius 0101101001110001?0?100012001?0111010000 “SO3” 010110101111000120110101200100111000011 “SO2” 010111?0????00022???0002200100111000000 Psilometriocnemus 102110?1011200122???1001000101111000110 Parachaetocladius 112110?1010010220???2111200101111020111 Limnophyes 110011?111120?0111010101100110101121111 Paralimnophyes 01011111011201020???0100100110101001011 Compterosmittia 111001100112001210110100200110101121111 Unniella 11011110011001021???0001111000000121?10 “MO5” 010111?00102010200100101200110111000010 Synorthocladius 01111110101201010000010000010000?000100 Heleniella ?11010?01112???02????001100101101021110

Table 6. Character states of pupae of Botryocladius species and outgroups. See Appendix 2 for character states.

5 10 15 20 “SO3” 10?00101111110000???? “SO2” 10?010000000000101100 B. grapeth 10?010001100000000012 B. tasmania 10?011122222000100012 B. brindabella 011100011111100001102 B. mdfrc 011100011111000001102 B. collessi 011100111111100001102 B. australoalpinus 10?000000000011102011 B. bibulman 110010022222000010012 B. petrophilus 110010122222000001112 B. freemani 10?010002221000102012 B. edwardsi 110010022222200002012 B. glacialis 1?0011122222200110002 B. mapuche 00?010000112010101001 B. tronador 00?010001112000101001