Propsilocerus Kieffer, 1923 shares morphological synapomorphies with Prodiamesinae

In their comprehensive analysis Cranston et al. (2012) quite unexpectedly recovered a monophyletic Orthocladiinae, except that Propsilocerus Kieffer, 1923, was recovered as a representative of Prodiamesinae. In this short communication I show that Propsilocerus shares morphological synapomorphies with other Prodiamesinae – namely (i) an indirect tracheal connection of the pupal thoracic horn to the adult spiracle and (ii) a parallel-sided rami of female gonapophysis IX. Introduction In their comprehensive analysis of molecular data, Cranston et al. (2012) quite unexpectedly recovered a monophyletic Orthocladiinae, despite the seeming lack of any morphological synapomorphies, but excluding Propsilocerus Kieffer, 1923. The placement of this genus, recovered as sister group to Prodiamesinae (Cranston et al. 2012), “can be reconciled on the basis of some morphology” listing large plates of the submentum, fringed anal lobes of the pupal abdomen and “and diverse volsellae in the male hypopygium”. However all these features, as Cranston et al. (2012) note, are, symplesiomorphies, and thus cannot corroborate the affinity of Propsilocerus to Prodiamesinae. Additionally, Propsilocerus lacks another common symplesiomorphy of the Prodiamesinae – the MCu crossvein. Cranston et al. (2012) note that absence of this character state in Propsilocerus renders morphological diagnosis of the Orthocladiinae even more problematic. It is also notable that Propsilocerus have only two precorneal setae while the rest of the Prodiamesinae for which immature stages are known, have three precorneals (Sæther 1986). Notably, Sæther (1986) had used the leg sheath arrangments and MCu presence and position in his key to the Prodiamesinae pupae, but not a type of the tracheal connection. Coffman (1979) had examined the connection between the pupal thoracic horn and the thoracic spiracle of the pharate adult as a possible phylogenetically informative character in Chironomidae. He found all examined representatives of Prodiamesinae (Monodiamesa Kieffer, 1922, Odontomesa Pagast, 1947, Prodiamesa Kieffer, 1906) have an indirect connection between the thoracic horn and a spiracle (Coffman 1979: p.43). This type of connection is represented by a bundle of the thin tracheoles originating from the spiracle and approaching close to the base of the thoracic horn, in contrast to the direct connection by a trachea in Podonominae and Tanypodinae, or the absence of such a connection, as in Orthocladiinae (Coffman 1979) (Figs 1A, B). Sæther (1977) noted that parallel rami of gonapophyses IX in the female genitalia could be Figure 1. A. Podochlus sp. pupa, habitus (brightfield microscopy); B. same, thoracic horn, tracheal connection to the spiracle is marked in blue (phase contrast). A


Introduction
In their comprehensive analysis of molecular data, Cranston et al. (2012) quite unexpectedly recovered a monophyletic Orthocladiinae, despite the seeming lack of any morphological synapomorphies, but excluding Propsilocerus Kieffer, 1923. The placement of this genus, recovered as sister group to Prodiamesinae (Cranston et al. 2012), "can be reconciled on the basis of some morphology" listing large plates of the submentum, fringed anal lobes of the pupal abdomen and "and diverse volsellae in the male hypopygium". However all these features, as Cranston et al. (2012) note, are, symplesiomorphies, and thus cannot corroborate the affinity of Propsilocerus to Prodiamesinae.
Additionally, Propsilocerus lacks another common symplesiomorphy of the Prodiamesinae -the MCu crossvein. Cranston et al. (2012) note that absence of this character state in Propsilocerus renders morphological diagnosis of the Orthocladiinae even more problematic. It is also notable that Propsilocerus have only two precorneal setae while the rest of the Prodiamesinae for which immature stages are known, have three precorneals (Saether 1986). Notably, Saether (1986) had used the leg sheath arrangments and MCu presence and position in his key to the Prodiamesinae pupae, but not a type of the tracheal connection. Coffman (1979) had examined the connection between the pupal thoracic horn and the thoracic spiracle of the pharate adult as a possible phylogenetically informative character in Chironomidae. He found all examined representatives of Prodiamesinae (Monodiamesa Kieffer, 1922, Odontomesa Pagast, 1947, Prodiamesa Kieffer, 1906 have an indirect connection between the thoracic horn and a spiracle (Coffman 1979: p.43). This type of connection is represented by a bundle of the thin tracheoles originating from the spiracle and approaching close to the base of the thoracic horn, in contrast to the direct connection by a trachea in Podonominae and Tanypodinae, or the absence of such a connection, as in Orthocladiinae (Coffman 1979) (Figs 1A, B). Saether (1977) noted that parallel rami of gonapophyses IX in the female genitalia could be A considered as synapomorphic for the Prodiamesinae (also true for Compteromesa Saether, 1981, for which only adults are known (Saether 1985)).
In this short communication I examine the condition of the abovementioned characters in Propsilocerus, to assist in elucidation of its phylogenetic position.

Materials and methods
The following material was examined from the ethanol and slide collection of Zoologische Staatssammlung München (ZSM): P. olivacea from "Bäm", Canada balsam mounted pupal exuviae, Thienemann collection, no collection date (as "Prodiamesa praecox"). Material was documented on a VHX-6000 digital microscope, following standard procedures (e.g. Haug et al. 2011). Ring light illumination was used with white background. Each image was recorded as a composite image combining images ("frames") of different focal plains ("z-stack") and several adjacent images to form a large panorama; processing was performed with the built-in software. Images were additionally recorded with several exposure times (HDR; Haug et al. 2011). Additionally, for the documentation of finer details, such as tracheal connections used BZX-900 fluorescent microscope, observing specimens using a brightfield, phase contrast microscopy as well as TRIC fluorescence (Haug et al. 2011). Individual photos subsequently stacked into the sharp composite using PICOLAY open software (www.picolay.de).  In the female genitalia, the paired rami of the gonapophyses IX of P. lacustris were distinctly parallel, similarly to examined P. olivacea and in accordance with the literature concerning other Prodiamesinae (Saether 1977: fig. 35.). It is notable that rami in Propsilocerus appear more weakly sclerotized than in other Prodiamesinae (Figs 5A-D, Saether 1977: fig. 35.).

Discussion
Examination shows that Propsilocerus is resolved not only as an ingroup-Prodiamesinae based on the multigene phylogeny of Cranston et al. (2012) but also shares important morphological synapomorphies with Prodiamesinae, thus further cementing location of Propsilocerus within this monophyletic subfamily.  Status of the Propsilocerus as an ingroup-Prodiamesinae sheds light on the persistence of morphological analysis in the age of molecular systematics. It also shows the danger of implicit definition of the groups by plesiomorphies. For example, in most fossil Prodiamesinae (I.e. Cretadiamesa Veltz, Azar et Nel, 2007, Lebanodiamesa Veltz, Azar et Nel, 2007 their purported affinities with Prodiamesinae appears based on plesiomorphies such as the presence and location of veins R 2+3 and MCu, rather than any meaningful synapomorphies (Veltz et al. 2007, Baranov et al. 2019. By defining fossil Prodiamesinae using such characters, we are in danger of including in Orthocladiinae some fossil representatives of Prodiamesinae that lack crossvein MCu. The fact that Orthocladiinae (as of now) still lack morphological synapomorphies (Saether 1977;Cranston 2000;Cranston et al. 2012) confuses the matter. Future studies of extant material through all the ontogenetic stages, as well as fossils, may eventually resolve this problem, and help us to locate morphological support for the unexpected Orthocladiinae monophyly (Cranston et al. 2012).

Acknowledgments
I am grateful to Martin Spies (ZSM Munich) for his invaluable help with the ZSM Diptera collection. I am also grateful to Peter Cranston (ANU Canberra) for the extensive discussion surrounding this note. Contribution is dedicated to William P. Coffman and Ole A. Saether, whose supreme skills as morphologists and attention to minute details still inspires us to go forward after their passing. I am grateful to Torbjørn Ekrem and anonymous reviewer for improving the manuscript.