The morphology of the eggs in the Hermanella complex (Ephemeroptera: Leptophlebiidae)

The Hermanella complex is a well-established monophyletic group of Hagenulinae mayflies, but the phylogenetic relationships among its members are still being discussed. In order to fill some gaps in the taxonomy of this group we describe, for the first time, the eggs of eight species of this complex (Hermanella amere, Hermanella mazama, Hydromastodon sallesi, Hydrosmilodon gilliesae, Hydrosmilodon plagatus, Leentvaaria palpalis, Paramaka incognita, Paramaka sp.) using scanning electron microscopy, including some important members whose generic allocation have been questioned. The egg morphology in these eight species of Hermanella complex and their similarities with other species of the complex are discussed. A R T I C L E I N F O Article history: Received 09 December 2020 Accepted 26 February 2021 Available online 26 March 2021 Associate Editor: Juliano Morimoto


Introduction
The mayflies of the Hermanella complex, or Hermanellognatha, form a monophyletic clade of Hagenulinae (Ephemeroptera, Leptophlebiidae) with nymphs showing specialized filtering mouthparts, such as enlarged labrum, and rows of long setae on maxillae and labium (Domínguez and Flowers, 1989;Kluge, 2007). These insects have a wide distribution in the Neotropical region, with some representatives extending to the Nearctic region (Flowers and Domínguez, 1991).
In a cladistic analysis performed by Domínguez and Cuezzo (2002), based on 5 genera and 11 OTU's (operational taxonomic units), egg characters were included in order to test their value to the analysis. The genera Leentvaaria and Paramaka were not included, while Hydromastodon was not described by that time. In this study, the monophyly of the Hermanella complex was supported with new synapomorphies, including chorion sculptures, showing that egg characters are important in phylogenetic studies for the group.
Considering that egg features are useful to understand the phylogeny of the Hermanella complex, data from additional species and genera may be useful to fill some gaps in future cladistics analysis. This study describes the chorion sculpture in the eggs of eight species from the Hermanella complex belonging to five genera, including Leentvaaria, Paramaka, and Hydromastodon.

Materials & Methods
The insects (preserved in 80% ethanol) were obtained from collection kept in the Museu de Entomologia da Universidade Federal de Viçosa (UFVB), Brazil and identified with aid of taxonomic keys and comparison with original descriptions (Polegatto and Batista, 2007;Lima et al., 2012;Nascimento and Salles, 2013;Domínguez et al., 2014;Salles et al., 2016). The eggs were removed from the abdomen of mature nymphs or adult females of eight species from five genera. The eggs were transferred to 90% ethanol for 15 min, hexamethyldisilazane for 10 min and air dried. Then the samples were fixed onto aluminum stubs, gold covered (20 nm thickness) and analyzed with a LEO VP1430 Scanning Electron Microscope at 15 kV in Núcleo de Microscopia e Microanálise, Universidade Federal de Viçosa (UFV). Terminology used for egg descriptions follows Koss and Edmunds (1974).

Figure 1
Hermanella amere a: General shape of egg. b: Knob-terminated coiled threads (arrow) concentrated only on the concave polar region. Hermanella mazama c: Chorionic surface smooth and general shape prismatic of the egg with concave and convex (arrowhead) polar region. d: Knob-terminated coiled threads (arrow) restricted to the concave polar region. Hydromastodon sallesi e: General shape, with knob-terminated coiled threads (arrows) distributed ring-like in the middle egg region and regularly distributed on the concave polar region. Hydrosmilodon gilliesae f: General shape of egg with knob-terminated coiled threads (arrow) regularly distributed on the surface. Scale bars: 10 µm.
Egg size: 120-136 µm in length, 67-124 µm in width. General shape prismatic, with one polar region convex, the other concave (Fig. 1e). Chorion surface smooth or with very fine small granules. KCTs distributed along a ring at the equatorial region and scattered in the on polar concave surface (Fig. 1e) Hydrosmilodon gilliesae Thomas & Péru, 2004in Thomas et al. (2004 (Fig. 1f).

Discussion
The general morphology of the eggs of the Hermanella complex was initially investigated by Domínguez and Flowers (1989), and since then some characteristics have been often encountered, such as polar regions flattened or slightly concave, terminal knobs of KCTs elevated above coiled thread, and a supraequatorial micropyle (Domínguez and Cuezzo 2002). The most of these characteristics are also present in the species here analyzed.
The eggs of He. amere and He. mazama with KCTs restricted to the polar egg region are similar to those found in Hermanella thelma Needham & Murphy and Hermanella froehlichi Ferreira & Domínguez (Figs. 1b and 3a in Domínguez and Cuezzo 2002). However, this feature is not exclusive for the genus because it also occurs in Hylister plaumanni Domínguez & Flowers (Domínguez and Flowers 1989), Paramaka and Hydromastodon (present study).
The general pattern of the eggs of L. palpalis with KCTs regularly distributed on the whole egg surface and prismatic shape are also found in species of other genera, for example He. guttata Domínguez & Flowers, N. ehrhardti (Ulmer), Hs. saltensis Flowers & Domínguez (Domínguez and Cuezzo 2002), and Hs. gilliesae (present study). In phylogenetic studies Leentvaaria has been claimed as the sister group of Needhamella ehrhardti (Domínguez et al., 2001) or Hs. gilliesae (Sartori, 2005). Salles et al. (2016) stated that some characteristics of the imago of L. palpalis are also found in Hs. gilliesae, suggesting that they are probably closely related, which is also suggested by the similar eggs features found here.
The chorion sculpture in the eggs of Hs. gilliesae is also similar to that found in Hs. saltensis, except the occurrence of granulated bumps in the later (Domínguez and Cuezzo, 2002). The egg morphology of Hs. plagatus is different from the general pattern found in other Hydrosmilodon species, and even in the Hermanella complex, what gives additional evidence to the non-monophyletic nature of Hydrosmilodon (see Salles et al., 2016).
We also presented the first egg description for Paramaka (P. sp. and P. incognita), and for Hydromastodon (Hm. sallesi). The eggs of these species are similar, with circular-shape, hexagonal in cross section, one polar region concave with KCTs and the other convex, smooth. Paramaka incognita and Hm. sallesi have also KCT distributed along an equatorial ring, a characteristic not found in any other representative of the Hermanella complex. Further studies are still necessary to verify if these are closely related genera.
This study describes for the first time the eggs of Leentvaaria, Paramaka, Hydromastodon and of additional species in Hydrosmilodon and Hermanella. Overall, our findings provide new data with potential to enhance the knowledge about the systematics of the Hermanella complex.