New insights into the phylogeny of Tortopus Needham and Murphy and Tortopsis Molineri (Ephemeroptera, Polymitarcyidae) with description of three new species

The family Polymitarcyidae, with a worldwide distribution, includes mayflies with large burrowing nymphs. South America harbors the highest diversity of this family, including the subfamilies Asthenopodinae and Campsurinae. In this work, three new species belonging to the genera Tortopsis and Tortopus (Campsurinae) are described based on adults and nymphs from Colombia: Tortopsis toro sp. nov., Tortopsis andaki sp. nov. and Tortopus coreguaje sp. nov. Additionally, Tortopsis limoncocha is firstly recorded from Colombia. A cladistic analysis of all the species in these genera is presented, using external morphological characters of adults and eggs. Keys to male and female adults of all the species of both genera are presented.


Introduction
The monophyletic group formed by Tortopus Needham and Murphy, 1924 and Tortopsis Molineri, 2010 (Campsurinae) gathers very specialized mayflies with burrowing nymphs and extremely short-lived adults (Domínguez et al. 2006). Nymphs present many morphological traits related to their digging habits and food filtering (McCaf-and a South American origin has been hypothesized for these groups (McCafferty 1998;Molineri et al. 2019).
The role of some Campsurinae species in bioturbation of lentic habitats, with large nutrient and pollutant movements from the sediment by the nymphs had received some attention (Leal et al. 2007). On the contrary, their role in the erosion of the clay-banks of large rivers has not been studied yet, but it may be as important as the effect of the related genus Asthenopus in degrading woody debris (Sattler 1967). Another little known aspect in this group is their silk production and use (Sattler 1967;Molineri and Emmerich 2010).
Tortopus was established by Needham and Murphy in 1924 and seven species are currently recognized (Molineri 2010; Molineri et al. 2012): T. arenales Molineri, T. bellus Lugo-Ortiz and McCafferty, T. circumfluus Ulmer, T. harrisi Traver, T. igaranus Needham and Murphy, T. pixuna Molineri, Boldrini and Salles and T. zottai (Navás). In Colombia, only T. igaranus, the type species of the genus, has been recorded from male and female imagos collected in the Peruvian-Colombian border on the Putumayo River (Needham and Murphy 1924;Molineri 2010). Tortopus is characterized by male genitalia with flattened and basally fused penis and ninth abdominal sternum with medio-longitudinal notch, short parastyli, female sternum VIII with long furrows anterior to sockets (parastyli receptors), and nymphs with two subapical tubercles on inner margin of mandibular tusks (Molineri 2010).
Tortopsis was established by Molineri (2010) for a group of species previously classified in Tortopus. Tortopsis is characterized by male genitalia with cylindrical penis, each penean arm separated from the base; ninth abdominal sternum entire, long curved parastyli present; female fore wing without additional veins between R 2 and IR, female sternum VIII with parastyli receptors C or V-shaped, and nymphs with a single subapical tubercle on mandibular tusks (Molineri 2010). Ten species are known from central Argentina to the northern United States of America (Molineri 2010;Gonçalves et al. 2011;Molineri et al. 2012): T. bruchianus (Navás), T. canum Gonçalves, Da Silva and Nessimian, T. limoncocha Molineri, T. obscuripennis (Domínguez), T. parishi (Banks), T. primus (McDunnough), T. puella (Pictet), T. sarae (Domínguez), T. spatula Molineri and T. unguiculatus (Ulmer). In Colombia, the genus is represented by only two species (T. unguiculatus and T. spatula) from several localities in the Amazonas department (Molineri 2010;Molineri et al. 2012). Molineri (2010) performed a phylogenetic revision of all the species in Tortopus and Tortopsis, but the taxonomic knowledge has been growing, especially during the last decade, with the description of new species and stages (Gonçalves et al. 2011;Molineri et al. 2012).The aim of the present work is to describe two new species of Tortopsis and a new species of Tortopus recently collected in a biologically poorly known area in Colombia. Additionally, we amend the phylogeny proposed in Molineri (2010), including all the species presently known. We also present keys to all the species of both genera.

Study area
The Caquetá River is located in the Amazon region, in the southeastern portion of Colombia and northwestern portion of Brazil. It extends through 2,280 km, with 1,200 km within Colombia and the remaining within Brazil. The river arises in the Colombian Massif known as "Páramo de las Papas", in the central Andean region of the Cauca department, and flows into the Amazon River in Brazilian territory, where it changes the name to Japurá. It has an extensive water network, and along with the Putumayo River, are the two main tributaries of the Colombian Amazon. The Caquetá River drains a large basin of approximately 267,730 km 2 , shared between the departments of Cauca, Caquetá, Putumayo, Guaviare, Vaupés and Amazonas within Colombia (IGAC 1999). Average flow rate ranges from a minimum of 231.8 m 3 .sec -1 (December-March) and a maximum of 1,298 m 3 .sec -1 (May-August), with a medium value of 417.1 m 3 .sec -1 , as recorded for a multi-year average flow in the Andaquiri Hydrological Station (IDEAM 2020). From a limnological point of view, it is a river of white water, due to its Andean origin. It is characterized by high electrolyte content and a significant load of suspended particles from the erosive processes that occur in the surrounding mountain range (Duque et al. 1997). Continuous navigation is very difficult due to the existence of many rapids and riffles along its course, particularly those within the Araracuara region, where the river runs through a deep and long canyon.
The type locality of the collected specimens here described corresponds to the lowlands of the middle basin of the Caquetá River, in the town of Curillo, located within the limits of the dense Amazon jungle in the department of Caquetá. All the material described here was collected at a single station in the town of Curillo, based on authorization 1166 granted by ANLA (National Authority of Environmental Licenses in Colombia) in the project "Trans-Amazon Aquatic Insects: study of potential areas of endemism in Colombia".

Collection and descriptions
Nymphs were manually collected on riverbanks, with the help of a blade to separate substrate pieces with the nymphal burrows. Substrate consisted of hard clay, and the pieces picked up were disaggregated on the shore to collect the nymphs. Mature nymphs were settled in rearing cages in the river for a short period and then transported in an ice keeper with river water and a piece of substrate (at ambient temperature). Adults were collected with a light trap at river margin set around sunset. All the collected material was fixed and preserved in ethyl alcohol 96°. Slide mountings were done using Canada Balsam. Photographs were taken with a ZEISS Axio-Cam ICc 5 mounted on a Stemi 508 stereo microscope and a Leica M205C stereomicroscope with an attached Leica MC-170HD camera. Line drawings were made using a camera lucida mounted on a microscope Olympus BX51. Some photographs are the result of stalking partially focused images with the software CombineZP (Hadley 2010

Phylogenetic analysis
The matrix and characters proposed by Molineri (2010) were amended including four new characters (Appendix 1), the three new species here described, as well as Tortopsis canum Gonçalves et al. (2011) and the information provided in Molineri et al. (2012) for Tortopus igaranus and Tortopus ipixuna. The matrix analyzed here ( Table  1) includes 26 taxa and 31 morphological characters. All the species of Tortopus and Tortopsis were included, together with six outgroups. One of these outgroups (Ephoron) was used to root the tree. Searches were conducted in TNT (Goloboff et al. 2008) under parsimony, with implied weights (with different k values, from 3 to 20). Group support was calculated using the symmetric Jackknifing function of TNT, this function repeatedly resamples the character matrix and compares the resultant trees with the shortest tree (we performed 250 resamplings) (Pol and Goloboff 2020). Three poorly known taxa were deactivated during some searches: Tortopus circumfluus, Tortopsis bruchianus, and Tortopsis parishi.
Diagnosis. Tortopsis toro sp. nov., known from imagos of both sexes and nymphs, can be distinguished from all other species of the genus by: In adults, 1) forewing length ranges between 12.2-13.2 mm (male) and 15.5-17.0 mm (female); 2) hyaline wings, with brownish veins and costal margin shaded slightly gray; 3) parastyli almost straight in lateral view, slightly curved medially, with a longitudinal ventral furrow (19)(20)(21); 4) penis apically widened and flattened, with rounded semicircular apical spine (Fig. 20); 5) female parastyli receptors with rounded and elongated sockets (Fig. 22); 6) head shaded black among ocelli, occiput with very light gray anastomosed lines (Figs 1, 6); 7) coloration as in Fig. 1. The nymphs can be separated from the others of the genus by: 1) mandibles at most with 2 stout small spines on inner margin of tusk (basally to large subdistal tubercle, see arrow in Fig. 24); 2) outer margin of tusk, just before distal spur, with a marked indentation (arrow in Fig. 25); 3) head shaded with gray on occiput as in Fig. 6; 4) developing fore wing buds pigmented with gray slightly along costal margin and base of longitudinal veins (similar to T. puella in Molineri 2008: figure 16).
Egg. Suboval. Length, 410-420 μm; width, 320-340 μm. Etymology. This species is dedicated to Dr. Beatriz Toro Restrepo, Universidad de Caldas, in recognition of her work in environmental education, friendship and contributions in fieldwork with aquatic insects of Colombia.
Male imago. Length (mm): body, 10.8-11.0; fore wing, 9.2-9.6; hind wing, 4.0; foreleg, 4.9-5.0; cerci, 25.0-26.0. General coloration whitish shaded dorsally with light purplish gray (Figs 27). Head. Whitish shaded with light brownish gray among ocelli and on scape and pedicel, flagellum hyaline. Thorax. Pronotum hyaline shaded with purplish gray dorsally as in Fig. 27. Mesonotum whitish yellow shaded with purplish gray along medioparapsidal sutures and contiguous area, medial zone paler. Metanotum whitish yellow shaded gray dorsally. Thoracic pleura and sterna whitish. Legs whitish shaded with light purplish gray on fore tibia and fore tarsi. Wings: Membrane hyaline very lightly tinted with purplish gray, longitudinal and cross veins purplish gray. Abdomen. Translucent white, shaded slightly but extensively with purplish gray on terga, including medial area; some markings are darker (Fig. 27) and a thin medial dark line is present along most terga; terga VIII-X shaded with darker gray. Abdominal sterna whitish; sternum IX shaded very slightly with gray on median area. Genitalia (Figs 28-29, 33-38): whitish except apex of parastylus yellowish; parastylus curved dorsally more markedly on apical third (in lateral view, Figs 28 and 34-35) and with a longitudinal ventral furrow along its entire length (Fig.  33); forceps and penis translucent white, shaded very slightly gray on forceps; penis slender, spine at apex of penis rounded and flattened . Caudal filament whitish translucent.
Etymology. This species is dedicated to the Andaki indigenous people, an American ethnic group that inhabited the upper Caquetá River basin.
Distribution. Known only from the type locality.  LG Dias, MC Zúñiga, B Toro, JP Chaux and C Molineri cols. Paratypes:
Diagnosis. Tortopus coreguaje sp. nov., known from male imago and nymph, can be distinguished from all other species of the genus by the following combination of charac-ters: In male imagos, 1) fore wing length 10.9-11.0 mm (male); 2) pale wings, veins translucent gray; 3) pedestal short with relatively large, straight and very long parastyli (Figs 56-57, 67 and 69) (parastylus 2.6 times the length of pedestal base, in lateral view the parastylus reach 0.37 of the length of forceps from base); 3) ventral knob on forceps base relatively small; 4) penis relatively thin at base (fused portion), not strongly expanded distally (Figs 56 and 67); and 5) relatively dark species, head shaded stronger between lateral ocelli, occiput shaded gray except on pale medial line (Fig. 65). In the nymph: 1) inner protuberances on mandibular tusk almost contiguous (Figs 59-61); 2) frontal tuft of setae with irregular sinuous form (arrow in Fig. 58); 3) ventral and dorsal projection at the apex of fore tarsus subequal in length (Fig. 63).  Male imago. Length (mm): body, 11.0; fore wing, 10.9-11.0; hind wing, 4.8; cerci, 28.0. General coloration yellowish white shaded with gray (Fig. 65). Head. Coloration yellowish white shaded widely with gray on dorsum, shaded stronger between lateral ocelli, except around median ocellus and hind margin (Fig. 65). Antennae: scape and pedicel yellowish white completely shaded with gray, flagellum hyaline. Thorax. Pronotum with anterior ring hyaline, shaded with black posterolaterally (Fig. 65); posterior ring shaded gray except on sublateral areas and medial line. Mesonotum whitish yellow shaded with gray along medioparapsidal sutures and shaded with black on a V-shaped mark between posterior scutal protuberances (Fig. 65). Metanotum whitish yellow shaded gray medially. Thoracic pleura and sterna whitish yellow, with grayish small marks medially on furcasterna. Legs yellowish white shaded with grayish (fore legs broken off and lost). Wings: Membrane hyaline except basally slightly grayish, longitudinal and cross veins translucent shaded slightly with gray. Abdomen. Abdominal segments translucent-hyaline. Tergum I shaded gray anterolaterally (Fig. 65), terga II-IX shaded more extensively towards rear segments, except on: medial line of terga II-VII, and two pale marks (one submedian and one sublateral) in II-IX; tergum X shaded more completely with gray. Abdominal sterna shaded widely with brownish gray except on intersegmental membranes and around gill sclerites (Fig. 66). Genitalia (Figs 56-57, 67-69): sternum IX shaded with black on posterior margin and at both sides of the median furrow; relatively large parastyli orangeish, straight and long (Fig. 56); parastylus 2.6 times the length of pedestal base, in lateral view the parastylus reach 0.37 of the length of forceps from base; forceps and penis yellowish white, shaded gray on forceps. Caudal filament whitish translucent.
Male nymph (immature). Length: body, 9.5 mm; cerci, 2.5 mm; caudal filament, 2.0 mm. General coloration whitish shaded with gray (Fig. 70). Head. Head shaded with gray in net-shaped pattern posteriorly to epicranial suture (Fig. 70); anteriorly to this suture with two small lateral irregular-sinuous-shaped tufts of short setae (anterior to lateral ocelli, arrow in Fig. 58); frontal ridge relatively straight in dorsal view (Fig. 58); fronto-clypeal region acutely projected medially (Fig. 58); a tuft of ca. 10 long setae basally to antennal condyle; and a group of 3 long setae anterior to eye and 6 long curved setae posterior to eye (Fig. 58). Mandibular tusks straight (Figs 59-61, 70), with 2 subdistal rigid inner setae and ca. 12 weaker but long setae forming a basal arc (Figs 59-61); inner margin with two large and contiguous tubercles, the subdistal directed ventrally and the submedian directed medially (Figs 59-61), also a small blade-like projection present basally to distal spur (arrow in Fig. 61). Maxillae with small triangular ventral gill. Hypopharynx and labium whitish. Thorax. Pronotum with narrow anterior ring (0.28 of total length of pronotum); antero-lateral corners acutely projected; shaded with black laterally on anterior ring and medially on posterior ring. Meso and metanotum shaded black medially except medial line; wingbuds whitish shaded gray only at base of costal area. Legs. Whitish with yellowish setae and sclerotized portions. Foreleg with tibia-tarsus flattened, remnants of the suture between tarsus and tibia visible, tarsal dorso-distal projection 0.5 of total length of claw (Figs 62-63); ventral surface of fore tibia with rows of long filtering setae as in Fig. 62; fore femur with anterobasal short U-shaped row of filtering setae and an immediately posterior small group of ca. 7 long rigid and simple setae; apex of fore tarsus with ventral and dorsal projections subequal in length (but the ventral is much thinner, Fig. 63). Middle leg with long setae on anterior and posterior (functionally ventral and dorsal, respectively) margins of femur, anterior margin of tibia and tarsus; apical third of tibia and tarsus completely covered with strong setae, apex of tibia with a brush of thick setae ventrally. Hind leg with short strong setae on posterior margin, and transverse subdistal row of short setae on dorsal surface; hind tibia and tarsus with long setae on posterior margin, anterior margin covered with short and strong setae. All tarsal claws slender and curved, without denticles. Abdomen (Fig.  70). Gill I single, small and elongated, remaining gills well developed and double. Terga II-IX with medio-longitudinal row of setae; abdominal sterna with lateral margins strongly covered with setae, increasing in number posteriorly, sterna V-VI also with row of shorter setae on posterior margin; sternum IX with a row of few long setae on posterior half, along medial line. Cerci with rows of setae at each article, mainly on basal fourth; terminal filament much thinner and with whorls of setae almost on its entire length.
Etymology. This species is dedicated to the Coreguaje community inhabiting along the piemont and lower parts of the Caquetá River and its affluents.
Distribution. Known only from the type locality.

Phylogenetic relationships
Searches including all the taxa (under both, equal and implied weights) resulted in many possible resolutions with their strict consensus showing a large politomy that included all the species in Tortopus and Tortopsis. To study the relationship among species in Tortopus and Tortopsis, we deactivated three of them (Tortopus circumfluus, Tortopsis bruchianus and Tortopsis parishi). These species present many missing entries in the matrix, as they are poorly known from their original descriptions and were not collected again. When deactivated, a unique and more resolved tree is found under implied weights (Fig.  71), with high support for the sister relationship between Tortopus and Tortopsis, and for each genus as separate monophyletic groups. Equal weights found both genera as monophyletic but relationships inside them were poorly resolved. Equal and implied weighting strategies found the same synapomorphies for the group Tortopsis + Tortopus, and for each genus, as detailed below. Synapomorphies defining the clade Tortopus + Tortopsis: Female wing veins thickened (character 2:1); female hind wing with anastomosed anal sector (character 3:1); female parastyli receptors on abdominal sternum VIII, formed by paired sockets (character 5:2); female parastyli receptors, Ushaped (character 6:1, later change to other forms); legs of imagos of both sexes (except male forelegs), reduced and distorted (character 9:1); male genitalia with two segments on forceps (character 11:1); with outer projected pedestals, forming a dorsal and pointed parastylus (character 13:3); knob at forceps base present (character 27:1); and egg without polar caps (character 30:0).

Taxonomy
The form of the penis in T. toro sp. nov. is similar to T. obscuripennis and T. puella, because of the abrupt distal widening. On the contrary, the semicircular spine at the apex of penis is similar to T. spatula, as well as the presence of a furrow along the parastyli (this last feature also shared with T. andaki sp. nov. The nymph of T. toro sp.nov. is similar to T. obscuripennis in the shape and setation of tusks, but the subapical outer indentation on the tusks of T. toro sp. nov. is more marked (Figs 6,10,25) if compared with the other species (Molineri 2010: figure 96). Additionally, T. obscuripennis presents much darker wingbuds, with more extensive gray shading on developing veins (Molineri 2008).
Tortopsis andaki sp. nov. is similar to T. spatula, but can be readily distinguished because T. andaki sp. nov. presents a more complex penis apex, with a rounded membranous lobe and two sclerotized projections (Figs  36-37). The nymph of T. spatula is unknown and the nymph here attributed to T. andaki sp. nov. has 7 stout small spines on the inner margin of the tusk and wingbuds completely whitish, the finding of the nymph of T. spatula will allow further comparisons between these species.
Tortopsis limoncocha was previously known from a single locality in the Ecuadorian portion of the Amazonas basin (Molineri 2010) and another locality in Brazilian Amazon (Molineri et al. 2012), so here is firstly recorded for Colombia.
The nymph here described for Tortopus coreguaje sp. nov. is the second nymph known for the genus; the nymph of T. harrisi ) presents the inner protuberances on mandibular tusk more separated (by a distance subequal to tusk width at this zone), the frontal tuft of setae is rounded in outline, and the fore tarsal distal projections are of different length (the ventral is much shorter). The male imago of T. coreguaje sp. nov. is similar to T. igaranus, because of the long and slender parastyli, nevertheless T. coreguaje sp. nov. is a larger and darker species, the penis are wider and the parastyli are longer (see key).

Phylogenetic relationships
Our results improved the resolution reached in the last published phylogenetic hypothesis (Molineri 2010), by adding six more species and four characters. Also, the few sister species pairs obtained by Molineri (2010), were not recovered again. Other interesting relationships were recovered instead, that are sustained on the evolution of some characters in male genitalia. For example, in Tortopus, the extreme shortening of the parastylus defines the politomy containing three species (T. ipixuna, T. harrisi, T. bellus). On the other hand, it was surprising that the extremely wide penis present in three species of Tortopsis (T. toro sp. nov., T. obscuripennis and T. puella) was not recovered as a synapomorphy for these three species. We expect that future knowledge of the nymphs of the species known only from adults will allow the inclusion of numerous new characters, but for the moment, this was not possible.

Conclusions
The biodiversity study in the Caquetá region (Figs. 72-75) has been restricted for approximately 50 years, since many locations remained inaccessible because of public order problems. This fieldwork was carried out precisely in the post-conflict period in Colombia, and shows the richness of the region and the need to expand the knowledge of its biodiversity as the first step for the implementation of future conservation and management proposals.

File 1
Authors: Molineri C, Dias LG, Zúñiga MC (2021) Data type: .pdf Explanation note: Matrix, list of characters and states (modified from Molineri 2010), and list of synapomorphies defining the unique shortest tree (Fig. 71) found under implied weighting parsimony..

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/asp.79.e62735.suppl1