Philibaetis gen. nov., a new genus from the Philippines (Ephemeroptera, Baetidae)

Investigations of type material and new material from the Philippines (Luzon) revealed that Baetis luzonensis Müller-Liebenau, 1982 and B. realonae Müller-Liebenau, 1982 do not belong to Baetis Leach, 1815. A new genus, Philibaetis gen. nov., is described to accommodate both species and both are re-described based on larvae. The new genus is characterised by having a rectangular labrum with a submarginal row of long, simple setae on the dorsal surface and ventrally on lateral margins long, simple, spine-like setae, on anterolateral margins long, feathered setae and medially long, bifid setae and a partial, submarginal row of lanceolate setae. Both mandibles have blade-like incisors and dorsally, a mediolateral patch of long, spine-like setae; additionally, the left mandible has a tuft of long, partly branched setae at the base of the subtriangular process. Philibaetis gen. nov. is further characterised by a hypopharynx with a medial tuft of stout setae and anterolaterally, two smaller tufts of stout setae, a galea-lacinia with the distal denti-seta tooth-like and directed against canines, a fore femur apically with stout setae, both on anterior and posterior side and without a femoral patch and a claw with one row of denticles and two or three subapical setae. The protogonostyli under the cuticle of male last instar larvae are folded in the Labiobaetis type, excluding their affiliation to the genus Baetis. COI sequences were obtained from both species. The genetic distance (Kimura 2-parameter) between them is 17.5% on average. Very limited genetic distances of 0% to 3% (0.75% on average) were found between specimens of P. luzonensis comb. nov.


Introduction
The family Baetidae has the highest species diversity amongst mayflies, comprising ca. 1,100 species in 114 genera (Sartori and Brittain 2015, Jacobus et al. 2019, Cruz et al. 2020, Kluge 2020, which is close to one third of all mayfly species worldwide. They have a cosmopolitan distribution, except in Antarctica and New Zealand. Investigations of the molecular phylogeny of the order Ephemeroptera revealed the relatively-primitive status of the family (Ogden and Whiting 2005, Ogden et al. 2009, Ogden et al. 2019. The generic diversity of Baetidae is the highest in the Afrotropical realm (ca. 40 genera), followed by the Neotropical (ca. 27 genera) and Oriental (ca. 28 genera) realms and finally the Nearctic (20 genera), Palaearctic (17 genera) and the Australasian (ca. 12 genera) realms (updated from Gattolliat and Nieto 2009). The number for the Oriental realm still seems to be rather low, taking into account that several regions with potentially high diversities (e.g. the Indian subcontinent, Indonesia incl. Borneo and the Philippines) belong to this realm. The last new genera of Baetidae from the Oriental region were described in 2020 from Indonesia and the Philippines (Kaltenbach et al. 2020a) and from Thailand (Suttinun et al. 2020).
The Philippines are a complex archipelago with more than 7500 islands, spanning the Asian-Australian faunal zone interface directly at the Wallace Line. Its extraordinary biodiversity was presumably supported by the complex biogeographic history and isolation of the archipelago, including landmass movements, collisions between landmasses of different origin in Miocene and temporary Pleistocene land bridges which were possible colonisation pathways of species, but also by environmental gradients along steep volcanic slopes (Brown andDiesmos 2010, Freitag et al. 2016). Recent data suggest that even biogeographic regions, previously categorised as one single unit (e.g. Greater Luzon), are composed of distinct centres of endemism that correlate with tectonic features (Vallejo 2014), further explaining high endemism and niche specialisation of species found in the country.
Here we describe a new genus from the Philippines, based on the type material of Baetis luzonensis Müller-Liebenau, 1982 andB. realonae Müller-Liebenau, 1982, as well as larvae collected in 1997 and 2019 in four different locations in the Philippines (Luzon). Further collections of Ephemeroptera and especially of Baetidae in the Philippines and other parts of Southeast Asia will unveil more unknown genera and many more species in the future.

Materials and methods
The new material was collected in 1997 and 2019 in four different locations in the Philippines (Luzon). Many more locations were sampled in this period without further findings of one of the two species (Kaltenbach et al. 2020b: figs 48, 49). The specimens were preserved in 70%-96% ethanol. The dissection of larvae was done in Cellosolve (2-ethoxyethanol) with subsequent mounting on slides with Euparal liquid, using an Olympus SZX7 stereomicroscope.
The DNA of part of the specimens was extracted using non-destructive methods allowing subsequent morphological analysis (see Vuataz et al. 2011 for details). We amplified a 658 bp fragment of the mitochondrial gene cytochrome oxidase subunit 1 (COI) using the primers LCO 1490 and HCO 2198 (Folmer et al. 1994, see Kaltenbach and Gattolliat 2020 for details). Sequencing was undertaken with Sanger's method (Sanger et al. 1977). The genetic variability between specimens was estimated using Kimura-2-parameter distances (K2P, Kimura 1980), calculated with the programme MEGA 7 (Kumar et al. 2016, http://www.megasoftware.net).
The GenBank accession numbers are given in Table 1, nomenclature of gene sequences, following Chakrabarty et al. (2013).
Drawings were made using an Olympus BX43 microscope. Photographs of larvae were taken using a Canon EOS 6D camera and the Visionary Digital Passport imaging system (http://www.duninc.com) and processed with Adobe Photoshop Lightroom (http://www.adobe.com) and Helicon Focus version 5.3 (http://www.heliconsoft. com). SEM pictures were taken using a FEI Quanta FEC 250 electron microscope (Thermo Fisher). Photographs were subsequently enhanced with Adobe Photoshop Elements 13.

Imagines. Unknown.
Etymology. Philibaetis is an arbitrary combination of letters with allusion to the Philippines, where the genus occurs and to the genus Baetis. The latter is with reference to the superficial similarities between both genera and the fact that the species of Philibaetis gen. nov. were up to now assigned to Baetis. Gender is masculine.
Labrum (Figs 1a-f, 6a, b). Rectangular, wider than long; on dorsal surface with submarginal row of long, simple setae; ventrally on lateral margin long, simple, spine-like setae, on anterolateral margin long, feathered setae and medially long, bifid setae and a partial, submarginal row of lanceolate setae.
Hypopharynx (Fig. 2a). With medial tuft of stout setae and anterolaterally two smaller tufts of stout setae.
Labium (Fig. 2d, e). Glossae basally broad, narrowing towards apex, shorter than paraglossae; apically and on both margins with long, spine-like setae; paraglossae truncate in distal part, on apex with three rows of long, robust, distally-pectinate setae and one stout, bifurcate seta in posterolateral area; labial palps segment II without protuberance, dorsally with row of spine-like setae near outer lateral margin.
Thorax. Hind protoptera (Fig. 4g). Absent. Foreleg . Femur with dense row of curved, spine-like setae on dorsal margin, on apex short, stout setae on anterior and posterior side; femoral patch absent; tibia with patellotibial suture, dorsal margin with row of medium, clavate setae; claw robust and pointed, with one row of denticles, 2-3 long subapical setae on posterior side and one short, thin subapical seta on anterior side.
Abdomen. . Seven pairs of gills on segments I-VII, dorsally orientated. Margin with minute denticles intercalating fine, simple setae.
Colouration (Fig. 5a-c). Head, thorax and abdomen dorsally brown, abdominal segments VI and IX brighter, fore protoptera brown. Head, thorax and abdomen ventrally light brown. Legs with femur and tibia light brown, with darker pattern as in Fig. 5a-c, tarsus brown. Caudalii light brown.
Labrum (Fig. 1a-f). Rectangular, length 0.6× maximum width. Distal margin with shallow medial emargination and small process. Dorsally with medium, fine, simple setae scattered over surface; submarginal arc of setae composed of one plus 6-7 long, simple setae. Ventrally with marginal row of setae composed of lateral long, simple setae, anterolateral long, feathered setae and medial long, bifid setae; medially additional, partial, submarginal row of fine, lanceolate setae. Ventral surface with ca. eight short, spine-like setae near lateral margin.
Left mandible (Fig. 1i-k, m). Incisor blade-like; kinetodontium with three denticles. Prostheca apically denticulate and with comb-shaped structure. Margin between prostheca and mola straight, with few minute denticles. Subtriangular process long and slender, above level of area between prostheca and mola. Basally of subtriangular process with tuft of long, partly-branched setae. Tuft of setae at apex of mola present.
Both mandibles with lateral margins convex. Dorsally with fine, simple setae scattered over basal surface and mediolaterally with patch of long, spine-like, simple setae.
Hypopharynx and superlinguae (Fig. 2a). Lingua longer than superlinguae, longer than wide; with well-developed medial tuft of stout setae, anterolaterally with two smaller tufts of stout setae. Superlinguae distally almost straight, lateral margins slightly rounded; long, fine, simple setae along distal margin.
Maxilla (Fig. 2b). Galea-lacinia ventrally with one simple, apical seta under canines. Inner dorsal row of setae with three denti-setae, distal denti-seta tooth-like and directed against canines, other denti-setae slender, bifid and pectinate. Medially with one spine-like seta and four medium to long, simple setae. Maxillary palp ca. 1.2× as long as length of galea-lacinia; palp segment II 1.8× length of segment I; short, fine, simple setae scattered over surface of segments I and II; apex of segment II distally constricted and pointed.
Labium (Fig. 2d, e). Glossa basally broad, narrowing towards apex, shorter than paraglossa; inner margin with three medium to long, spine-like, simple setae; outer margin with four long, spine-like, simple setae; apex with two long and one very short, spine-like setae; ventral surface with very few short, fine, simple setae. Paraglossa distally truncate, slightly curved inwards; ventrally with three rows of long, robust, distally-pectinate setae in apical area, row of long, simple setae on anterolateral margin, one short, simple seta in mediolateral area and one robust, bifurcate seta in posterolateral area; dorsally with one long, spine-like seta near inner margin. Labial palp with segment I 0.6× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II without protuberance; ventral surface with short, fine, simple setae; dorsally with row of ca. six spine-like setae near outer margin. Segment III almost semicircular; length 0.8× width; ventrally covered with short, spine-like setae and short to medium, fine, simple setae.
Thorax. Hind protoptera (Fig. 4g). Absent. Foreleg (Figs 3a-c, 11a-c). Ratio of fore femur:tibia:tarsus:claw 1.2:1.0:0.4:0.2. Femur. Rather broad, length ca. 3× maximum width. Dorsal margin with row of ca. 40 long, curved, spine-like setae; length of setae mostly ca. 0.2× maximum width of femur. Apex rounded, with several curved, spine-like setae and many short, stout setae; on posterior side, arc of short, stout setae. Stout, lanceolate setae scattered along ventral margin; femoral patch absent. Tibia. Dorsal margin with row of medium, clavate setae. Ventral margin with row of short, spine-like setae, on apex some longer, spine-like setae and tuft of fine, simple setae. Anterior surface scattered with short, stout, lanceolate setae. Patellotibial suture present on basal ¾ area. Tarsus. Dorsal margin with row of short, stout and fine, simple setae. Ventral margin with row of short to medium, curved, spine-like setae. Claw robust and distally pointed, with one row of ca. seven denticles; generally three and sometimes two long, subapical setae.
Abdomen. Tergites (Fig. 4h). Surface with irregular rows of U-shaped scale bases, scattered micropores and fine, simple setae along posterior margin. Posterior margin of tergite IV without spines. Gills (Fig. 4a-d). Seven pairs of gills on segments I-VII. Margin with minute denticles intercalating fine, simple setae. Tracheae extending from main trunk to inner and outer margins. Gill I as long as length of half segment II. Gill IV as long as length of segments V and half VI combined. Gill VII as long as length of segment VIII.
Paraproct (Fig. 4e, f). Without prolongation at posterior margin, with ca. 16 stout, marginal spines. Surface scattered with U-shaped scale bases and fine, simple setae; with a patch of notched scales. Cercotractor with small, marginal spines. Protogonostyli (Fig. 10a) in male, last instar larvae ready to moult folded under larval cuticle in the Labiobaetis type.

Re-description
Colouration (Fig. 9a-c). Head, thorax and abdomen dorsally brown, abdominal segments VI and IX brighter, fore protoptera brown. Head, thorax and abdomen ventrally light brown. Legs with femur and tibia light brown, with darker pattern as in Fig. 9a-c, tarsus brown. Caudalii light brown.
Labrum (Fig. 6a, b). Rectangular, length 0.6× maximum width. Distal margin with shallow medial emargination and small process. Dorsally with medium, fine, simple setae scattered over surface; submarginal arc of setae composed of one plus 6-7 long, simple setae. Ventrally with marginal row of setae composed of lateral long, simple setae, anterolateral long, feathered setae and medial long, bifid setae; medially an additional, partial, submarginal row of fine, lanceolate setae. Ventral surface with ca. eleven short, spine-like setae near lateral margin.
Left mandible (Fig. 6e-g). Incisor blade-like; kinetodontium with three denticles. Prostheca apically denticulate and with comb-shaped structure. Margin between prostheca and mola straight. Subtriangular process long and slender, above level of area between prostheca and mola. Basally of subtriangular process with tuft of long, partly-branched setae. Tuft of setae at apex of mola present.
Both mandibles with lateral margins convex. Dorsally with fine, simple setae scattered over basal surface and mediolaterally with patch of long, spine-like, simple setae.
Hypopharynx and superlinguae (Fig. 6h). Lingua longer than superlinguae, longer than wide; with well-developed medial tuft of stout setae, anterolaterally with two smaller tufts of stout setae. Superlinguae distally almost straight, lateral margins almost straight; long, fine, simple setae along distal margin.
Maxilla (Fig. 6i). Galea-lacinia ventrally with one simple, apical seta under canines. Inner dorsal row of setae with three denti-setae, distal denti-seta tooth-like and directed against canines, other denti-setae slender, bifid and pectinate. Medially with one spine-like seta and four medium to long, simple setae. Maxillary palp ca. 1.1× as long as length of galea-lacinia; 2-segmented; palp segment II 1.9× length of segment I; short, fine, simple setae scattered over surface of segments I and II; apex of segment II distally constricted and pointed.
Labium (Fig. 6j, k). Glossa basally broad, narrowing towards apex, shorter than paraglossa; inner margin with three short to long, spine-like, simple setae; outer margin with three long, spine-like, simple setae; apex with two long and one very short, spine-like setae; ventral surface with very few short, fine, simple setae. Paraglossa distally truncate, slightly curved inwards; ventrally with three rows of long, robust, distally pectinate setae in apical area, row of long, simple setae on anterolateral margin, one short and one robust, bifurcate seta in posterolateral area; dorsally with one long, spine-like seta near inner margin. Labial palp with segment I 0.7× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II without protuberance; ventral surface with short, fine, simple setae; dorsally with row of ca. five spine-like setae near outer margin. Segment III almost semicircular, apically slightly pointed; length 0.8× width; ventrally covered with short, spine-like setae and short to medium, fine, simple setae.
Thorax. Hind protoptera. Absent. Foreleg (Figs 7a-e, 11d, e). Ratio of fore femur:tibia:tarsus:claw 1.2:1.0:0.5:0.2. Femur. Rather broad, length ca. 3× maximum width. Dorsal margin with row of ca. 31  long, curved, spine-like setae; length of setae mostly ca. 0.2× maximum width of femur. Apex rounded, with two pairs of curved, spine-like setae and many short, stout setae; on posterior side, arc of short, stout setae. Stout, lanceolate setae scattered along ventral margin; femoral patch absent. Tibia. Dorsal margin with row of medium, clavate setae. Ventral margin with row of short, spine-like setae, on apex, some longer, spine-like setae and tuft of fine, simple setae. Anterior surface scattered with short, stout, lanceolate se-tae. Patellotibial suture present on basal ¾ area. Tarsus. Dorsal margin with some fine, simple setae. Ventral margin with row of short to medium, curved, spine-like setae. Claw robust and distally pointed, with one row of eight or nine denticles; with ca. three stripes; two long, subapical setae.
Abdomen. Tergites (Fig 7i). Surface with irregular rows of U-shaped scale bases and scattered micropores. Posterior margin of tergite IV with triangular spines, wider than long. Gills (Fig. 8a, b). Seven pairs of gills on segments I-VII. Margin with minute denticles intercalating fine, simple setae. Tracheae extending from main trunk to inner and outer margins. Gill I as long as length of ⅔ segment II. Gill IV as long as length of segments V and half VI combined. Gill VII little longer than length of segment VIII.
Protogonostyli (Fig. 10b) in male, last instar larvae ready to moult folded under larval cuticle in the Labiobaetis type.

Assignment, morphological differences and similarities
Philibaetis gen. nov. obviously belongs to the family Baetidae, based on the developing turbinate eyes of late instar male larvae, the long, slender body shape, the larval Y-shaped frontal suture reaching ventrally to lateral ocelli (Fig. 8c), the labrum with distinctly-expressed median incision, the shape of left and right prostheca, the kinetodontium fused with mandible and with incisor, the shape of glossae (basally widened, most part narrow) and the anterior outer projection of femur apex, which is directed towards inner side of femur (Figs 3a, 7a) (Wang and McCafferty 1996, Kluge 2004, Kluge and Novikova 2011. It further belongs to the subfamily Baetinae sensu Kazlauskas (1972) and the Anteropatellata, because of the presence of the patellotibial suture on the forelegs of the larvae (Kluge and Novikova 2011). It can be assigned to the non-Baetis complex of Baetinae (Waltz et al. 1994, Waltz andMcCafferty 1997), also referred to as Baetungulata-non-Baetofemorata (Kluge and Novikova 2011), because of the claw with one row of denticles on inner-anterior side and the lack of a femoral patch. The folding of the protogonostyli developing under the cuticle of last instar male larvae is of the Labiobaetis type ( Fig. 10; Kluge 2004: fig. 29E-J). Therefore, Philibaetis gen. nov. is preliminarily placed within the tribe Labiobaetini Kluge and Novikova (2016) since the final placement can be only definitely done once the male imago is described as well.
Apart from the unique combination of characters as described in the diagnosis, the following characters differentiate Philibaetis gen. nov. from all other genera of Baetinae: labrum ventrally on lateral margin with long, simple, spine-like setae, on anterolateral margin with long, feathered setae and medially with long, bifid setae and a partial submarginal row of lanceolate setae 6a,b); both mandibles with blade-like incisor and dorsally, a mediolateral patch of long, spinelike setae, left mandible additionally with a tuft of long, partly-branched setae at base of subtriangular process (Figs 1g-m, 6c-g); galea-lacinia with distal denti-seta tooth-like and directed against canines (Figs 2b, c, 6i); claws with one row of denticles and 2 or 3 long, subapical setae on posterior side and one reduced subapical seta on anterior side (Figs 3b, 7d, 11a-e).

Subapical setae of claws
Müller-Liebenau described both species of Philibaetis gen. nov. (but previously assigned to Baetis) with one subapical seta only (Müller-Liebenau 1982a: figs 2H, 3H). This can be explained by the fact that the subapical setae of this genus often appear as one single seta in slide preparations or under a binocular. However, our slides, as well as SEM pictures, revealed the number and complex structure of these subapical setae. P. luzonensis comb. nov. generally has three long subapical setae on the claws of all legs (posterior side) and P. realonae comb. nov. always two; additionally, there is always one reduced subapical seta on the anterior side in both species (Figs 3b, 7d, 11a-e). All subapical setae on the posterior side of the same claw have separate insertions, standing very close together. They seem to have different lengths and partly different shapes, which could facilitate the arrangement to one single, broader seta under natural conditions. The separation of these combined setae seems to happen under unnatural conditions by the influence of chemicals like alcohol and Cellosolve (2-ethoxyethanol) during slide preparation; or by dehydration during preparation for SEM pictures, but not always. Several long, subapical setae on the same side of a claw are very rare in Baetidae.  fig. 10) and Moribaetis McCafferty, 1985, with two (Lugo-Ortiz and fig. 9). One single, long subapical seta is widespread in Baetidae (e.g. Baetis, Baetodes Needham & Murphy, 1924, Gratia Thomas, 1992, Indobaetis Müller-Liebenau & Morihara, 1982 and one on each side was described from, for example, the different genera of the Centroptiloides complex, Madaechinopus Gattolliat & Jacobus, 2010, Offadens Lugo-Ortiz & McCafferty, 1998, Liebebiella Waltz & McCafferty, 1987, Monocentroptilum Kluge, 2018 as well as a few species of Baetis (Müller-Liebenau and Morihara 1982, Müller-Liebenau 1982b, Müller-Liebenau 1984, Thomas 1992, Lugo-Ortiz and McCafferty 1998, 1999. Claws and subapical setae, SEM photos: a-c. Philibaetis luzonensis comb. nov., a. Fore claw, anterior view; b. Middle claw, posterior view; c. Hind claw, posterior view; d, e. Philibaetis realonae comb. nov., d. Fore claw, anterior view; e. Middle claw, posterior view. Gattolliat 2002, Dominguez et al. 2006, Kluge 2018, Yanai et al. 2018). However, the subapical setae have variable positions on the claw between some of the genera.

Genetics
The interspecific genetic distance between the two species of Philibaetis gen. nov. is 17%-18% (K2P; Table 2). This is in line with interspecific distances reported from Labiobaetis in Indonesia (11%-24%), Borneo (19%-25%) and the Philippines (15%-27%) (Kaltenbach and Gattolliat 2019a, 2020, Kaltenbach et al. 2020b). Ball et al. (2005) reported a mean interspecific, congeneric distance of 18% for mayflies from the United States and Canada. The intraspecific distances for P. luzonensis comb. nov. are very low as expected, ranging from 0% to 3% (average 0.75%; K2P; Table 2). Distribution So far, both P. luzonensis comb. nov. and P. realonae comb. nov. were found on Luzon only, but at very different altitudes (Table 3, Fig. 12). This could be an indication that the two species were differentiating along elevational and environmental gradients, as discussed for some species of Labiobaetis on the same island (Kaltenbach et al. 2020b and citations therein).  The number of sampled localities and different habitats is still limited in the Philippines and there are large regions without any collection activities so far. Therefore, we may assume that the number of taxa will continue to increase with further collections in the future.