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The geographically widespread species Afrixalus laevis (Anura: Hyperoliidae) currently has a disjunct distribution in western Central Africa (Cameroon, Equatorial Guinea, Gabon, and possibly adjacent countries) and the area in and near the Albertine Rift in eastern Democratic Republic of the Congo and neighboring countries. At least two herpetologists have previously suggested that these disjunct populations represent distinct species, and herein, we utilize an integrative taxonomic approach with molecular and morphological data to reconcile the taxonomy of these spiny reed frogs. We sequenced 1554 base pairs of the 16S and RAG1 genes from 34 samples of A. laevis and one sample of A. orophilus (sympatric with eastern populations of A. laevis), and combined these data with previously sequenced GenBank Afrixalus samples via the bioinformatics toolkit SuperCRUNCH. Phylogenetic trees, dated phylogenetic analyses, and species-delimitation analyses were generated with RAxML, BEAST, and BPP, respectively. Eleven mensural characters were taken from multiple specimens of A. laevis and A. orophilus, and compared with paired t-tests and analyses of covariance. These combined results suggested populations of A. laevis in western Central Africa (Cameroon and Bioko Island, Equatorial Guinea) represent one species, whereas populations from the Albertine Rift and nearby forests represent two undescribed taxa that are sister to A. dorsimaculatus. The two new species (A. lacustris sp. nov. and A. phantasma sp. nov.) are distinguished by our phylogenetic and species-delimitation analyses, significant differences in several mensural characters, qualitative morphological differences, and by their non-overlapping elevational distribution.

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Secondary sympatry amongst sister lineages is strongly associated with genetic and ecological divergence. This pattern suggests that for closely related species to coexist in secondary sympatry, they must accumulate differences in traits that mediate ecological and/or reproductive isolation. Here, we characterized inter- and intraspecific divergence in three giant tree frog species whose distributions stretch across West and Central Africa. Using genome-wide single-nucleotide polymorphism data, we demonstrated that species-level divergence coincides temporally and geographically with a period of large-scale forest fragmentation during the late Pliocene. Our environmental niche models further supported a dynamic history of climatic suitability and stability, and indicated that all three species occupy distinct environmental niches. We found modest morphological differentiation amongst the species with significant divergence in tympanum diameter and male advertisement call. In addition, we confirmed that two species occur in secondary sympatry in Central Africa but found no evidence of hybridization. These patterns support the hypothesis that cycles of genetic exchange and isolation across West and Central Africa have contributed to globally significant biodiversity. Furthermore, divergence in both ecology and reproductive traits appear to have played important roles in maintaining distinct lineages. At the intraspecific level, we found that climatic refugia, precipitation gradients, marine incursions, and potentially riverine barriers generated phylogeographic structure throughout the Pleistocene and into the Holocene. Further studies examining phenotypic divergence and secondary contact amongst these geographically structured populations may demonstrate how smaller scale and more recent biogeographic barriers contribute to regional diversification.La sympatrie secondaire parmi les espèces sœurs est fortement associée à la divergence génétique et écologique. Ce modèle suggère que pour que des espèces étroitement liées coexistent en sympatrie secondaire, elles doivent accumuler des différences dans les traits qui contribuent à l'isolement écologique ou reproductif. Ici, nous avons caractérisé la divergence inter- et intra-spécifique chez trois espèces de grenouilles arboricoles géantes dont les distributions s'étendent à travers l'Afrique de l'Ouest et Centrale. Avec des données génétiques, nous avons démontré que la divergence au niveau des espèces coïncide temporellement et géographiquement avec une période de fragmentation forestière à la fin du Pliocène. Nos modèles de niches environnementales ont soutenu une histoire dynamique de stabilité climatique, et ont indiqué que les trois espèces occupent des niches environnementales distinctes. Nous avons trouvé une différenciation morphologique modeste parmi les trois espèces mais une divergence significative dans le diamètre du tympan et les cris des mâles. De plus, nous avons confirmé que deux espèces sont présentes en sympatrie secondaire en Afrique Centrale mais n'avons trouvé aucune preuve d'hybridation. Ces résultats soutiennent l'hypothèse que les cycles d'échange génétique et d'isolement à travers l'Afrique de l'Ouest et Centrale ont contribué à une profonde concentration de biodiversité dans la région. De plus, la divergence des traits écologiques et reproducteurs semble avoir joué un rôle important dans le maintien de lignées distinctes. Au niveau intra-spécifique, nous avons constaté que les refuges climatiques, les gradients de précipitation, les incursions marines et potentiellement les barrières fluviales ont généré une structure phylogéographique pendant le Pléistocène et jusqu'à l'Holocène. Des études examinant la divergence phénotypique et le contact secondaire entre ces populations géographiquement structurées pourraient démontrer comment des barrières biogéographiques à échelle plus petite et plus récentes contribuent à la diversification régionale.

Afrixalus lacustris Greenbaum, Dehling, Kusamba & Portik sp. nov. Great Lakes Spiny Reed Frog urn:lsid:zoobank.org:act: 41D3C41A-4501-4BEC-BFB6-B9C6C1518DC3 Afrixalus laevis (nec Megalixalus laevis Ahl 1930)— Laurent 1950: 24; Laurent 1972: 59 (partim); Laurent 1982: 33 (partim); Schiøtz 1999: 56 (partim); Channing & Howell 2006: 137 (partim); Spawls et al. 2006: 183 (partim); Channing & Rödel 2019: 158 (partim). Holotype. UTEP 20805 (field no. ELI 605), adult male, from the vicinity of Kalundu (03.15552° S, 28.42108° E, 1482 m), South Kivu Province, DRC, collected on 21 December 2010 by Chifundera Kusamba and Félix I. Alonda (Figs. 8D–F, 12A–B). Paratopotype. UTEP 20806 (field no. ELI 606), adult male, collected with the holotype. Referred specimens. Specimens with an asterisk were not included in morphological analyses due to poor quality of preservation, lack of a voucher, or unavailability of specimen(s): UTEP 20807–08 (field nos. ELI 644, 649), vicinity of Kalundu (03.15552° S, 28.42108° E, 1482 m), South Kivu Province, DRC; UTEP 20809, 22422 (field nos. ELI 669–70), Baraka, near shore of Lake Tanganyika (04.10832° S, 29.09705° E, 777 m), South Kivu Province, DRC; UTEP 20810–11 (field nos. EBG 1316, 1319), vicinity of Irangi (01.8873° S, 28.4495° E, 820 m), South Kivu Province, DRC; UTEP 22423 (field no. MUSE 10192), Kahuzi-Biega National Park, Nanwa (02.362138° S, 28.192589° E, 1566 m), South Kivu Province, DRC; UTEP 22424 (field no. MUSE 10137), Itombwe Plateau, Mbandakila (03.47937° S, 28.413049° E, 1135 m), South Kivu Province, DRC; RMCA 77-020 -B-133–136* (four specimens), Itombwe Plateau, Tubutubu (2300 m), South Kivu Province, DRC; RMCA 77-020 -B-89–92 (field nos. MF 14–15 [two specimens]), Kitutu (ca. 03.28° S, 28.11° E, 700 m), South Kivu Province, DRC; RMCA 77-020 - B-0069–71 (1 specimen), Itula (ca. 03.27° S, 28.12° E, 650 m), South Kivu Province, DRC; RMCA 77-020 -B-0081–83, 77-20-B-0086, Kamituga (ca. 03.54° S, 27.69° E, 1050 m), South Kivu Province, DRC; RMCA 77-020 - B-0072–73 (1 specimen), Mwana (ca. 03.15° S, 28.47° E, 1650 m), South Kivu Province, DRC; UTEP 22417 (field no. CRSN 2773), Toyokana (02.02734° N, 030.06653° E, 1294 m), Ituri Province, DRC; (field no. EPLU 395 *, photo and tissue only), ca. 0.5 km E of Epulu, near Mt. Mbiya (01.39594° N, 28.58093° E, 755 m), Ituri Province, DRC; UTEP 22416 (field no. DFH 247), Bwindi Impenetrable National Park, Buhoma (00.99045° S, 29.61884° E, 1523 m), Western Region, Uganda; (field nos. DFH 1102–03 * photos and tissues only), Kibale Forest National Park, Ngogo Research Center (00.49795° N, 30.42262° E, 1363 m), Western Region, Uganda; CAS 202032–36, Bwindi Impenetrable National Park, Buhoma rd., 2 km S (by rd.) of Bizenga River (01.00975° S, 29.620694° E), Western Region, Uganda; CAS 202133, Bwindi Impenetrable National Park, small tributary to Ishasha River (00.905° S, 29.704972° E, 1280 m), Western Region, Uganda; CAS 202109, Bwindi Impenetrable National Park, Bizenga River at Buhoma rd. (00.99275° S, 29.615722° E), Western Region, Uganda; CAS 256035 *, Bwindi Impenetrable National Park, rd. north of Ruhija, Western Region, Uganda; CAS 256128–31 *, Mabira Forest Reserve, marshy area adjacent to unpolluted stream (0.4508° N, 32.9493° E, 1121 m), Mukono District, Central Region, Uganda. Diagnosis. The species is referred to the genus Afrixalus for exhibiting the following characteristics: fingers and toes webbed; tips of fingers and toes enlarged to discs; eye large; pupil vertically elliptical; tympanum indistinct; vomer ridges and teeth absent; dorsal surfaces finely shagreened with minute pointed tubercles; outer metatarsal tubercle distinct; gular gland in males. It is readily distinguished from most other members of the genus by its small size (SVL in males 18.9–22.2 mm, in females 20.8–25.7 mm), being smaller than A. dorsalis (males 25–28 mm, females 26–30 mm), A. fornasini (males 30–38 mm, females 35–40 mm), A. fulvovittatus (males 23–27 mm, females 25–28 mm), A. lacteus (males 22–27 mm, females 25–29 mm), A. leucostictus (males 27–32 mm, females 25–36 mm), A. manengubensis (males to 32 mm), A. nigeriensis (males 28–34 mm, females 32–35 mm), A. osorioi (males 27–31, females 32–35 mm), A. paradorsalis (males 28–34 mm, females 32–35 mm), A. septentrionalis (males 19–21 mm), A. vittiger (males 22–25 mm, females 25–28 mm), and A. wittei (males 27–30 mm, females 29–33 mm); and larger size than A. delicatus (males 15–19 mm, females 17–22 mm) and A. stuhlmanni (males 15–21 mm, females 17–25 mm). Its differs in dorsal coloration and pattern from all species with longitudinal stripes or bands on a yellowish-brown background (A. brachycnemis, A. crotalus, A. delicatus, A. dorsalis, A. enseticola, A. fornasini, A. fulvovittatus, A. knysnae, A. morerei, A. orophilus, A. quadrivittatus, A. schneideri, A. spinifrons, A. stuhlmanni, A. upembae, A. septentrionalis, A. vittiger, and A. wittei); species showing a conspicuous large dark dorsal blotch on a uniform background on anterior dorsum in combination with large blotches on both sides of the hip (A. leucostictus, A. manengubensis, A. osorioi, A. paradorsalis, and A. schneideri) or with longitudinal lateral dark bands (A. equatorialis and A. nigeriensis); species with a uniformly colored dorsum without conspicuous pattern except a dark dorsolateral stripe (A. aureus, A. clarkei, A. delicatus, A. fornasini, A. lacteus, A. leucostictus [dorsum with small white tubercles], A. uluguruensis, and A. weidholzi [might have a black vertebral line and dark brown flanks]); and from all remaining species having either dark stripes running from tip of snout to back, crossing each other and continuing as dorsolateral stripes (A. vibekensis, also males without asperities); anterior part of dorsum yellowish with brown pattern, posterior part of dorsum translucent (A. laevis, also males without asperities); posterior part of dorsum and dorsal sides of limbs semi-translucent with dense small dark white-edged speckles (A. dorsimaculatus); and dark blotches and transverse marks with white speckles (A. sylvaticus). It is most similar to A. phantasma sp. nov. but differs from this species in being smaller (Table 1) and showing an overall darker coloration and more contrasting dorsal pattern that is obvious even in preserved specimens (Figs. 7–8, 12). It is readily distinguished from A. phantasma sp. nov. by the more extensive hand and toe webbing (Fig. 8E–F). Description of holotype. Measurements of the holotype are provided in Table 2. Body very slender, widest at temporal region, slightly tapering to groin (Fig. 8D–F); head small (HL/SVL 0.31, HW/SVL 0.31), about as long as wide (HW/HL 0.99); snout relatively long, rounded in dorsal view and in lateral profile, slightly wider than long; canthus rostralis hardly discernible, slightly concave between eye and nostril in dorsal view; loreal region oblique; nostrils rounded, directed anterolaterally and slightly dorsally; situated much closer to tip of snout and to eye, separated from each other by distance about equal to distance between eye and nostril (IN/EN 1.02); eye directed anterolaterally, strongly protruding, very large (ED/HL 0.39), its diameter shorter than snout (ED/SL 0.90); interorbital distance much wider than upper eyelid (IO/EW 1.61) and wider than internarial distance (IO/IN 1.31); tympanum covered by skin, not visible externally; upper jaw with dentition; teeth on premaxilla larger than teeth on maxilla; choanae small, located far anterolaterally at margins of roof of mouth, its anterior edge covered by maxillary bone, therefore appearing semicircular in ventral view; vomer ridges and teeth absent; tongue heartshaped, longer than broad, bilobed for about one-fourth of its length, free distally for about two-thirds its length; densely covered with minute papillae; median lingual process absent. Dorsal surfaces of head, trunk, and limbs and lateral surfaces of trunk finely shagreened; tiny pointed, widely scattered tubercles on dorsum, more numerous posteriorly than anteriorly; supratympanic fold absent; few tubercles at rear end of mandible without pointed tips; ventral side of head smooth; vocal sac present; gular gland of vocal sac smooth, large and wide, covering about 70 percent of throat width (Fig. 8E); chest smooth, abdomen weakly areolate; ventral side of limbs smooth; short transverse fold above vent. Forelimbs slender; hand large (HaL/SVL 0.28); tips of fingers enlarged into large disks, each with circummarginal groove; relative length of fingers: I I 2-/2 II 2-/3- III 2.25/2 IV; thenar tubercle oval, small and very low, about one-fourth length of metacarpal of Finger I; inner palmar tubercle and outer palmar tubercle very low, small, rounded, almost indiscernible. Hind limbs slender, moderately long; heel reaching to level of eye when legs adpressed forwardly to body; crus moderately long (TibL/SVL 0.44), slightly shorter than thigh; heels not touching each other when knees flexed and thighs held perpendicularly to median plane; foot subequal in length to crus (FoL/TibL 0.94); relative length of toes: I I 1.5/2+ II 1+/2+ III 1+/2+ IV 2/1+ V (Fig. 8E); inner metatarsal tubercle moderately prominent, elongated, about half length of metatarsus of Toe I; outer metatarsal tubercle small, rounded. Coloration in life. During night, skin on dorsal side of head, trunk, forelimbs, crus and tarsus light brown with more or less regular, locally more densely clustered dark brown speckling; distinct dark brown transverse stripe between upper eyelids, another less distinct one in scapula region, and another narrow one at level of anterior end of pelvis; dorsal skin of thigh largely unpigmented with only a broad stripe of widely scattered brown pigmentation; distinct dark brown band along canthus, continuing behind eye on both sides of trunk to level of pelvis; tubercles below eye and in tympanic region at rear end of jaw white; dark brown stripe running diagonally on middle of tibia, forming interrupted, inverted U-shaped band together with pelvic spots when legs folded against body; skin of ventral side of head, trunk and limbs largely unpigmented and translucent; gular gland bright yellow; fingers and toes yellow (Fig. 12). During day, basic dorsal coloration light brown; dark brown dorsal speckles, spots and stripes in more pronounced contrast to basic coloration, more distinctly visible. Coloration in preservative. Dorsal basic coloration largely faded to light brown; darker dorsal pattern elements dark brown, clearly visible; color of gular gland, fingers and toes faded to white (Fig. 8D–F). Variation. The paratypes match the holotype in general appearance and proportions. Coloration and color pattern varies from light-cream to yellowish-orange with a few relatively large and darker blotches and vermiculation, light speckling and indistinct dorsolateral stripe to an almost reticulated pattern of contrasting bright and dark elements with a broad and conspicuous brown dorsolateral band (Fig. 12). Pedal webbing variation is I 1.5[100]/2+[100] II 1+[100]/2+[100] III 1 +[50],1.25[50]/2+[100] IV 2 [100]/1+[100] V. Bioacoustics. The advertisement call and other vocalizations are unknown. Ecology and natural history. We collected males and females in swamps in forest openings and near forest edges (Fig. 11B), from vegetation at the edge of streams (or on vegetation overhanging them) in forest, and from non-forest vegetation near the shore of Lake Tanganyika. Egg deposition has not been observed and tadpoles are unknown. An adult male (CAS 256035) was found in Bwindi Impenetrable National Park (Uganda) ca. 2 m above ground in a tree fern “either calling or moving to a calling site (similar to ‘running’ of Kassina)” (D. Blackburn, in litt., 22 July 2021). Laurent (1955) noted the species from transitional forest. In his paper on amphibians of Virunga National Park, Laurent (1972) noted the species is most commonly found in swamps with herbaceous vegetation in secondary forest, and more rarely, in primary forest. Laurent (1982) noted that, in general, the species is common in the foliage of shrubs in dense forests. He also recorded it from “une mare sombre” (i.e., a dark pond) in syntopy with Chiromantis at Kitutu (DRC), marshes where it reproduces by sticking its eggs under the leaves of fountain grass (Pennisetum, Shabunda, DRC), a shaded bank of a pond in syntopy with Hyperolius “ tuberculatus ” (likely H. hutsebauti sensu Bell et al. 2017) and H. frontalis; the center of this pond had reeds harboring H. kivuensis and Afrixalus orophilus (Shabunda, DRC). Laurent (1983:352) listed the species (along with Phrynobatrachus petropedetoides and Hyperolius frontalis) from “shadowy puddles of the thick transition forest.” Etymology. The species epithet is the Latin adjective “ lacustris,” meaning belonging to or dwelling in lakes; in allusion to the distribution of the new species in the region of the African Great Lakes. Distribution and conservation. The species is distributed from lowland rainforest of the Congo Basin at 460 m (Omaniundu, DRC, Laurent 1982) to transitional forests of the Albertine Rift at 1650 m (Mwana, DRC) and east as far as Mabira Forest, Uganda (1121 m). As shown in Figure 5, the species has a relatively large distribution, including several national parks and protected areas (e.g., Virunga National Park, Bwindi Impenetrable National Park), and thus is not likely to be threatened based on a limited distribution. Using GeoCAT, its extent of occurrence is estimated as 409,238 km ² and its area of occupancy as 160 km ² (Bachman et al. 2011). We therefore classify this species as Least Concern according to the IUCN Red List criteria (IUCN 2021). Four specimens (RMCA 77-020-B-133–136) from Tubutubu, Itombwe Plateau (2300 m) are problematic because they are morphologically consistent with A. lacustris, but they were collected from a substantially higher elevation than all other known specimens. Coordinates for Tubutubu provided by RMCA (4° S, 28.933° E) suggest the locality is east of the highest point of the plateau at an elevation of about 1400 m on Google Earth. Although it is possible that the specimens were collected at the latter elevation, Laurent (1982) mentions bamboo forest as the habitat, which usually occurs at the highest elevations of the Itombwe Plateau (Doumenge 1998). No clarity is offered by Laurent (1964), because he does not mention any Afrixalus in his study of the ecology and distribution of amphibians of the Itombwe Plateau. Given this conflicting information, we do not include the Tubutubu specimens in our current understanding of the elevational distribution of A. lacustris (see gray triangle in Fig. 5)., Published as part of Greenbaum, Eli, Portik, Daniel M., Allen, Kaitlin E., Vaughan, Eugene R., Badjedjea, Gabriel, Barej, Michael F., Behangana, Mathias, Conkey, Nancy, Dumbo, Bonny, Gonwouo, Legrand N., Hirschfeld, Mareike, Hughes, Daniel F., Igunzi, Félix, Kusamba, Chifundera, Lukwago, Wilber, Masudi, Franck M., Penner, Johannes, Reyes, Jesús M., Rödel, Mark-Oliver, Roelke, Corey E., Romero, Soraya & Dehling, J. Maximilian, 2022, Systematics of the Central African Spiny Reed Frog Afrixalus laevis (Anura Hyperoliidae), with the description of two new species from the Albertine Rift, pp. 201-232 in Zootaxa 5174 (3) on pages 218-223, DOI: 10.11646/zootaxa.5174.3.1, http://zenodo.org/record/6986207, {"references":["Ahl, E. (1930) Ueber die afrikanischen Arten der Baumfroschgattung Megalixalus. Sitzungsberichte der Gesellschaft Naturforschender Freunde zu Berlin, 1930, 89 - 102.","Laurent, R. (1950) Genres Afrixalus et Hyperolius (Amphibia Salientia). Exploration du Parc National Albert, Mission G. F. De Witte (1933 - 1935), 64, 5 - 120, 7 pls.","Laurent, R. F. (1972) Amphibiens. Exploration du Parc National des Virunga, Deuxieme Serie, 22, 1 - 125, 11 pls.","Laurent, R. F. (1982) Le genre Afrixalus Laurent (Hyperoliidae) en Afrique Centrale. Annales du Musee Royal de l'Afrique Centrale, Serie in Octavo, Science Zoologique, 235, 1 - 93.","Schiotz, A. (1999) Treefrogs of Africa. Edition Chimaira, Frankfurt am Main, 350 pp.","Channing, A. & Howell, K. M. (2006) Amphibians of East Africa. Cornell University Press, Ithaca, New York, New York, xi + 418 pp.","Spawls, S., Howell, K. M. & Drewes, R. C. (2006) Pocket Guide to the Reptiles and Amphibians of East Africa. A & C Black Publishers, London, 240 pp.","Channing, A. & Rodel, M. - O. (2019) Field Guide to the Frogs & Other Amphibians of Africa. Struik Nature, Cape Town, 408 pp.","Laurent, R. F. (1955) Apercu de la biogeographie des batraciens et des reptiles de la region des grand lacs. Bulletin de la Societe Zoologique de France, 79, 290 - 310.","Bell, R. C., Parra, J. L., Badjedjea, G., Barej, M. F., Blackburn, D. C., Burger, M., Channing, A., Dehling, J. M., Greenbaum, E., Gvozdik, V., Kielgast, J., Kusamba, C., Lotters, S., McLaughlin, P. J., Nagy, Z. T., Rodel, M. - O., Portik, D. M., Stuart, B. L., VanDerWal, J. & Zamudio, K. R. (2017) Idiosyncratic responses to climate-driven forest fragmentation and marine incursions in reed frogs from Central Africa and the Gulf of Guinea Islands. Molecular Ecology, 26 (19), 5223 - 5244. https: // doi. org / 10.1111 / mec. 14260","Laurent, R. F. (1983) About the herpetofauna of Central African montane forest. In: Rhodin, A. G. J. & Miyata, K. (Eds.), Advances in Herpetology and Evolutionary Biology: Essays in Honor of Ernest E. Williams. Museum of Comparative Zoology, Cambridge, Massachusetts, pp. 350 - 358.","Bachman, S., Moat, J., Hill, A., de la Torre, J. & Scott, B. (2011) Supporting Red List threat assessments with GeoCAT: Geospatial Conservation Assessment Tool. ZooKeys, 150, 117 - 126. https: // doi. org / 10.3897 / zookeys. 150.2109","IUCN (2021) IUCN Red List Categories and Criteria: Version 2021 - 3. Available from: https: // www. iucnredlist. org / (accessed 25 May 2022)","Doumenge, C. (1998) Forest diversity, distribution, and dynamique in the Itombwe Mountains, South-Kivu, Congo Democratic Republic. Mountain Research and Development, 18 (3), 249 - 264. https: // doi. org / 10.2307 / 3674036","Laurent, R. F. (1964) Adaptive modifications in frogs of an isolated highland fauna in Central Africa. Evolution, 18 (3), 458 - 467. https: // doi. org / 10.1111 / j. 1558 - 5646.1964. tb 01622. x"]}

Afrixalus phantasma Dehling, Greenbaum, Kusamba & Portik sp. nov. Ghost Spiny Reed Frog urn:lsid:zoobank.org:act: 1B2DBF26-5B0A-4DA8-B65E-0BC01704C1B4 Afrixalus laevis (nec Megalixalus laevis Ahl 1930)— Laurent 1972: 59 (partim); Laurent 1982: 33 (partim); Schiøtz 1999: 56 (partim); Channing & Howell 2006: 137 (partim); Spawls et al. 2006: 183 (partim); Channing & Rödel 2019: 158 (partim). Holotype. ZFMK 103454 (field no. JMD 723), adult male, from Gishwati Forest (01.823745° S, 29.360373° E, 2084 m), now part of Gishwati-Mukura National Park, Western Province, Rwanda, collected on 5 April 2011 by J. Maximilian Dehling and Bonny Dumbo (Figs. 7A, 8A–C). Paratypes. ZFMK 103455 (field no. JMD 722), adult female, collected with the holotype; ZFMK 103456 (field no. JMD 2015-30), adult male, ZFMK 103457 (field no. JMD 2015-31), adult female, from the type locality in Gishwati Forest, both collected on 27 September 2015 by J. Maximilian Dehling and Bonny Dumbo; ZFMK 103458–60 (field nos. JMD 677–679), three adult males, collected on 19 March 2011 by J. Maximilian Dehling, ZFMK 103461–62 (field nos. JMD 954–955), two adult males, collected on 16 February 2013 by J. Maximilian Dehling, all from Kamiranzovu Swamp (02.477165° S, 29.158243° E, 1962 m), Nyungwe National Park, Western Province, Rwanda; UTEP 20791–20792 (field nos. EBG 2838, 2843), two adult males, collected on 21 December 2009 by Chifundera Kusamba, Wandege M. Muninga, Mwenebatu M. Aristote, and Maurice Luhumyo from Nyakasanza Swamp near Tshibati (02.22886° S, 28.78017° E, 1979 m), South Kivu Province, DRC. Referred specimens. UTEP 20802 (field no. EBG 1198), forest ca. 4 km NW of Lwiro (02.2226° S, 28.7754° E, 2077 m), South Kivu Province, DRC; UTEP 20803, 22418–20 (field nos. EBG 1232, 1238–40), Kahuzi-Biega National Park, Mugaba (02.2750° S, 28.6631° E, 2298 m), South Kivu Province, DRC; UTEP 20804 (field no. EBG 1283), Kahuzi-Biega National Park, Chinya (02.2671° S, 28.6455° E, 2267 m), South Kivu Province, DRC; UTEP 20793–94 (field nos. EBG 2844–45), Nyakasanza Swamp near Tshibati (02.22886° S, 28.78017° E, 1979 m), South Kivu Province, DRC; UTEP 20795–20800, 22421 (field nos. ELI 414–20), Nyakasanza Swamp near Tshibati (02.22829° S, 28.77972° E, 1979 m), South Kivu Province, DRC; UTEP 20801 (field no. ELI 425), Chanjoka (02.21261° S, 28.77644° E, 2115 m), South Kivu Province, DRC. Diagnosis. The species is referred to the genus Afrixalus by exhibiting the following characteristics: fingers and toes webbed; tips of fingers and toes enlarged to discs; eye large; pupil vertically elliptical; tympanum indistinct; vomer ridges and teeth absent; dorsal surfaces finely shagreened with minute pointed tubercles; outer metatarsal tubercle distinct; gular gland in males. It is readily distinguished from most other members of the genus by its small size (SVL in males 20.1–24.6 mm, in females 22.6–26.4 mm), being smaller than A. dorsalis (males 25–28 mm, females 26–30 mm), A. fornasini (males 30–38 mm, females 35–40 mm), A. lacteus (males 22–27 mm, females 25– 29 mm), A. leucostictus (males 27–32 mm, females 25–36 mm), A. manengubensis (males to 32 mm), A. nigeriensis (males 28–34 mm, females 32–35 mm), A. osorioi (males 27–31 mm, females 32–35 mm), A. paradorsalis (males 28–34 mm, females 32–35 mm), A. septentrionalis (males 19–21 mm), and A. wittei (males 27–30 mm, females 29–33 mm); and larger than A. brachycnemis (males 18–21 mm, females 20–22 mm), A. delicatus (males 15–19 mm, females 17–22 mm), A. spinifrons (males to 20 mm, females to 25 mm), and A. stuhlmanni (males 15–21 mm, females 17–25 mm). Its differs in dorsal coloration and pattern from all species with longitudinal stripes or bands on a yellowish-brown background (A. brachycnemis, A. crotalus, A. delicatus, A. dorsalis, A. enseticola, A. fornasini, A. fulvovittatus, A. knysnae, A. morerei, A. orophilus, A. quadrivittatus, A. schneideri, A. spinifrons, A. stuhlmanni, A. upembae, A. septentrionalis, A. vittiger, and A. wittei); species with a conspicuous large dark dorsal blotch on a uniform background on anterior dorsum in combination with large blotches on both sides of the hip (A. leucostictus, A. manengubensis, A. osorioi, A. paradorsalis, and A. schneideri) or with longitudinal lateral dark bands (A. equatorialis and A. nigeriensis); species with a uniformly colored dorsum without conspicuous pattern except a dark dorsolateral stripe (A. aureus, A. clarkei, A. delicatus, A. fornasini, A. lacteus, A. leucostictus [dorsum with small white tubercles], A. uluguruensis, and A. weidholzi [might have a black vertebral line and dark brown flanks]); and from all remaining species having either dark stripes running from tip of snout to back, crossing each other and continuing as dorsolateral stripes (A. vibekensis, also males without asperities); anterior part of dorsum yellowish with brown pattern, posterior part of dorsum translucent (A. laevis, also males without asperities); posterior part of dorsum and dorsal sides of limbs semi-translucent with dense small dark white-edged speckles (A. dorsimaculatus); and dark blotches and transverse marks with white speckles (A. sylvaticus). The advertisement calls of the following species of Afrixalus consist of a long series of clicks, often initiated by a long buzzing note, and thus differ from the call of A. phantasma: A. aureus, A. brachycnemis, A. clarkei, A. crotalus, A. delicatus, A. dorsalis, A. fornasini, A. fulvovittatus, A. knysnae, A. nigeriensis, A. morerei, A. osorioi, A. quadrivittatus, A. septentrionalis, A. spinifrons, A. vibekensis, A. vittiger, A. weidholzi, and A. wittei. The advertisement calls of the remaining species for which information is available differ from the call of A. phantasma in the following characteristics (in parentheses): A. dorsimaculatus (soft, short buzzing rattle, usually repeated three times), A. equatorialis (series of five notes, repeated at 15–20/s and initiated by a long buzz, energy maximum at 2000–2500 Hz), A. lacteus (8–9 notes, 14–15 pulses,>300 pulses/s), A. paradorsalis (2–3 notes, energy maximum at 2700 Hz), A. stuhlmanni (2–9 notes, energy maximum at 4200–5100 Hz), and A. sylvaticus (2–5 notes, energy maximum at 4000–4500 Hz). The new species is most similar to A. lacustris sp. nov. from which it differs by a number of characteristics (see below). Description of holotype. Measurements of the holotype are provided in Table 2. Body very slender, widest at temporal region, slightly tapering to groin (Fig. 8A–C); head small (HL/SVL 0.31, HW/SVL 0.30), about as long as wide (HW/HL 0.98); snout relatively long, rounded in dorsal view and in lateral profile, slightly longer than wide; canthus rostralis hardly distinct, straight between eye and nostril; loreal region oblique; nostrils rounded, directed anterodorsally and slightly laterally; situated much closer to tip of snout and to eye, separated from each other by distance larger than distance between eye and nostril (IN/EN 1.15); eye directed anterolaterally, strongly protruding, very large (ED/HL 0.41), its diameter shorter than snout (ED/SL 0.84); interorbital distance much wider than upper eyelid and wider than internarial distance (IO/IN 1.21); tympanum covered by skin, not visible externally; upper jaw with dentition; teeth on premaxilla larger than teeth on maxilla; choanae small, located far anterolaterally at margins of roof of mouth, its anterior edge covered by maxillary bone, therefore appearing semicircular in ventral view; vomer ridges and teeth absent; tongue short and moderately broad, bilobed for about one-sixth of its length, free distally for about half its length; densely covered with minute papillae; median lingual process absent. Dorsal surfaces of head, trunk, and limbs and lateral surfaces of trunk finely shagreened; minute pointed, widely scattered tubercles on dorsum, more numerous posteriorly than anteriorly; supratympanic fold absent; small, subcircular, elevated glandular area in tympanic region posterior to eye and rear end of mandible, bearing large tubercles with pointed keratinous tips; ventral side of head smooth; vocal sac present; gular gland of vocal sac smooth, large and wide, covering about 70 percent of throat width (Fig. 8B); chest smooth, abdomen weakly areolate; ventral side of limbs smooth; short transverse fold above vent. Forelimbs slender;hand large (HND/SVL 0.32); tips of fingers enlarged into large disks, each with circummarginal groove; relative length of fingers: I I 2+/2.25 II 2/3+ III 2.75/2.25 IV; thenar tubercle oval, small and low, about one-fourth length of metacarpal of Finger I; palmar tubercles indiscernible. Hind limbs slender, moderately long; heel reaching to level of eye when legs adpressed forwardly to body; crus moderately long (TibL/SVL 0.48), about as long as thigh; heels meeting each other when knees flexed and thighs held perpendicularly to median plane; foot subequal in length to crus (FoL/TibL 0.96); relative length of toes: I I 2/2.25 II 1.5/3- III 1.75/3 IV 3-/1.75 V (Fig. 8C); inner metatarsal tubercle moderately prominent, elongated, about twothirds length of metatarsus of Toe I; outer metatarsal tubercle small, rounded. Coloration in life. During night, skin on dorsal side of head, trunk, forelimbs, crus and tarsus light yellowishbrown with more or less regular dark brown speckling; indistinct darker brown transverse stripe between upper eyelids; large dark brown, irregularly shaped spot in scapula region, two less distinct, somewhat lighter brown, irregularly shaped spots at level of anterior end of pelvis, separated from each other by half their width; cloacal region with dark brown mottling; dorsal skin of thigh largely unpigmented with only a broad stripe of widely scattered brown pigmentation; weak brown stripe along canthus, continuing behind eye on anterior half of trunk on both sides; tubercles below eye, and in tympanic region at rear end of jaw white; indistinct dark brown stripe running diagonally on middle of tibia, forming interrupted, inverted U-shaped band together with pelvic spots when legs folded against body; ventral side of head, trunk and limbs largely unpigmented and translucent; gular gland bright yellow; fingers and toes yellow (Fig. 7A). During day, basic dorsal coloration brighter, very light brown to bright cream-colored; dark brown dorsal speckles, spots and stripes in more pronounced contrast to basic coloration, more distinctly visible (Fig. 7B). Coloration in preservative. Dorsal basic coloration largely faded to yellowish white; darker dorsal pattern elements light to dark brown, clearly visible; yellow color of gular gland, fingers and toes faded to white (Fig. 8A–C). Variation. The paratypes match the holotype in general appearance, proportions, coloration and color pattern. Mensural variation within the species is shown in Table 1. Pedal webbing variation is I 2+[100]/2.5[90],2.25[10] II 2[50],2+[40],2.25[10]/3[10],3+[40],3.5[50] III 1.75 [80],2-[20]/3[100] IV 3 -[40],3[60]/1.75[100] V. Bioacoustics. Series of advertisement calls of six different males were recorded at the following ambient temperatures: 10.9°C (N = 1), 13.6°C (N = 3), and 16.2°C (N = 2). The advertisement call consisted of five to six, rarely four pulse groups (notes) (Fig. 9). Depending on the ambient temperature, notes were repeated at a rate of 7.1–10.0/s (10.9°C), 8.2–11.7/s (13.6°C), and 10.2–11.4/s (16.2°C). The highest repetition rate was always between the first three notes of a series, the lowest at the end of the series. Each note consisted of 10–11 pulses (Fig. 9). Probably due to echo effects, pulsation was often veiled in the waveforms, especially towards the end of the note. Pulse repetition rate varied from 190–210/s at 10.9°C, 222–250/s at 13.6°C, and 277–292/s at 16.2°C, resulting in a note length of 72–94 ms, 55–70 ms, and 36–50 ms, respectively. Amplitude modulation was prominent within individual notes (Fig. 9). Total call length showed linear temperature dependence and varied from 388–397 ms at 16.2°C, 472–499 ms at 13.6°C, and 572–620 ms at 10.9°C for five-note calls (Fig. 10). Energy maximum showed linear temperature dependence, with 3020–3150 Hz at 10.9°C, 3370–3550 Hz at 13.6°C, and 3660–3810 Hz at 16.2°C (Figs. 9–10). There was no marked frequency modulation. Prominent harmonics were at about 6000–7000 Hz and 9000–11000 Hz (Fig. 9). Ecology and natural history. We collected males and females in swamps in forest openings, or at forest edges (Fig. 11A). Males were found calling above standing bodies of water with thick lower vegetation cover. In Gishwati Forest and Kamiranzovu Swamp in Nyungwe Forest, the species was found calling in syntopy with Hyperolius castaneus and H. discodactylus. One adult male (UTEP 20802) from Kahuzi-Biega National Park (DRC) was found 3.5 meters above ground in montane forest. Tadpoles are unknown. Laurent (1955, 1983) listed the species from bushes in montane forest. Laurent (1982) noted that, in general, the species is common in the foliage of shrubs in dense forests. He also recorded it in banana trees in a deep valley (Nyungwe, Rwanda) and “des trous de prospection” (i.e., prospecting holes likely for gold mining, Upper Lubitshako, Kabobo Plateau, DRC). Etymology. The species epithet derives from the Greek noun φάντασμα (phántasma), meaning ghost or phantom, in allusion to the coloration and general appearance of the new species. The epithet is used as an invariable noun in apposition. Distribution and conservation. The occurrence of the species has been confirmed for several locations in western Rwanda (Nyungwe and Gishwati Forest) and eastern DRC (in and near Kahuzi-Biega National Park, Itombwe Plateau and Kabobo Plateau [Laurent 1982], Fig. 5). We expect the species to be eventually found in the AR of southwestern Uganda and northwestern Burundi. So far, the species has been recorded from only a narrow elevation range between 1962 m (Kamiranzovu Swamp, Rwanda) and ca. 2400 m (May ya Moto [and possibly Luemba], Itombwe Plateau, Laurent 1982). Given the relatively limited overall geographic distribution of the species with an estimated extent of occurrence of 19,088 km 2 (Fig. 5), an estimated area of occupancy of 52 km 2 (both calculated with GeoCAT; Bachman et al. 2011), the detection of the amphibian chytrid fungus in one adult male (UTEP 20791, Greenbaum et al. 2015), and the conservation challenges facing natural areas of the AR (Greenbaum 2017; Ayebare et al. 2018), we categorize this species as Vulnerable under the IUCN Red List criteria (IUCN 2021).

This article presents an experimental arrangement which, using 3-D numerical modeling, aims to study biomedical effects using subnanosecond pulsed electric fields (PEFs). As part of a major effort into developing contactless technology, the final aim of this study is to determine conditions of the applied PEFs (number of pulses, strength, pulse repetition frequency) able to produce electropermeabilization. The arrangement uses a pulsed power generator producing voltage impulses with an amplitude of up to 20 kV on a 50- $\Omega $ matched load, with a rise time of 100 ps and a duration of 600 ps. During the preliminary study reported here, samples containing E. Coli were exposed to PEFs in a 4-mm standard electroporation cuvette, allowing the application of a peak electric field strength of up to 60 kV/cm. The studies were facilitated by detailed 3-D electromagnetic modeling of the electric field distribution generated by voltage impulses inside the system. Due to the nature of tests, the numerical analysis played an essential role in the interpretation of results. Preliminary biological results reported in this study are very encouraging, showing that trains of 5000 to 50 000 pulses applied at a pulsed repetition frequency of 200 Hz (maximum PRF) can efficiently induce E. Coli electropermeabilization.

International audience; B-cell lines and primary PBMCs are notoriously hard to transfect, thus making genome editing, ectopic gene expression, or gene silencing experiments particularly tedious. Here we propose a novel efficient and reproducible protocol for electrotransfection of lymphoblastoid, B-cell lymphoma, leukemia cell lines, and B cells from PBMCs. The proposed protocol requires neither costly equipment nor expensive reagents; it can be used with small or large plasmids. Transfection and viability rates of about 79% and 58%, respectively, have been routinely achieved by optimizing the salt concentration in the electrotransfection medium and the amount of plasmid used. A validation of the protocol was obtained via the generation of a TP53 −/− RPMI8866 lymphoblastoid cell line which should prove useful in future hematological and blood cancer studies.

The R script and the database associated with the 2019 Merged model for Madagascar.