Honoring the Afro-Colombian musical culture with the naming of Epipedobates currulao sp. nov. (Anura, Dendrobatidae), a frog from the Pacific rainforests

Ho nori ng th e Afro- Col om bia n m usi cal cultur e with th e n ami ng of E pip ed ob at es [to be 1 rev eal ed] s p. n ov. (A n ura: D en dro bati d ae), a fro g from th e P a cific rainfor est s 2 3 Mileidy B eta nco urth-C un d ar 1 , Juan Ca milo Ríos-Orj uel a 1,2 , Andr ew J. Cra wfor d 1,3 , David C. 4 Ca nn atell a 4 , Re becca D. Tarvin 5 5 6 1 Dep arta me nto de Cie ncias Biol ógicas, U niversida d d e los A nd es, Bo g otá, 1 11 7 11, Colo mbi a 7 2 Grup o d e M orfolo gía y Ecol ogí a Evol utiva, Instituto d e Ci encias N atur ales, U niversida d N acion al d e 8 Col om bia, S ed e B o got á, 74 9 5, Col om bia 9 3 Muse o d e Historia N atur al C.J. Marink elle, Universid a d d e los An d es, Bo got á, 1 11 71 1, C olom bi a 10 4 Dep artm ent of Int egr ative Bi olo gy an d Bi odiversity Ce nter, U niversity of Texas, Au stin, TX 7 87 12, 11 U SA 12 5 Muse um of Verte brat e Z ool ogy an d D ep artme nt of Inte grative Biol ogy, U niversity of Califor nia, 13 Berkel ey, Berkel ey, CA 9 4 72 0, US A 14 15 Ab stra ct 16 Th e n um ber of am p hibia n sp ecies d escribed ye arly shows n o sig ns of slowin g d o wn , especially in 17 tropical re gio ns, implying t hat th e bi odiver sity of amphi bia ns rem ains w oef ully un der estimate d. W e 18 describe a n ew sp ecies of p oiso n frog fro m the Pacific lowla n ds of south w estern C olo mbi a: 19 Epi pe do b ates [to b e reve ale d] sp. nov., n am ed for th e P acific music an d d ance ge n re kno wn as 20 ba mb uco viej o or [to b e reve ale d] . This sp ecies inh abits lo wla nd for ests from 0– 2 60 m. This taxo n 21 differs from con ge n ers by havin g a co mbi natio n of yell ow blotch es in the dors al ant erior regi o n of th e 22 thigh an d u p per ar m, h om og en o us dark-b row n d orsal colorati on, a n d a dvertisem ent calls of long 23 dur ation an d m a ny pulses. W e also describe th e co urtship call of E. [to be rev eal ed] , which is low er in 24 frequ e ncy an d shorter in dur atio n th an th e advertisem e nt call. Molec ular p hylo ge neti c analyses 25 confirm th e mo n op hyly of the p o pul atio ns sampl ed an d its positio n as the sister-gro up of 26 Epi pe do b ates n arin ensis , which occurs in south wester n C olo mbi a. Am o ng sp ecies o f Epip ed o bat es , 27 the n e w species h as b ee n pr eviously conf used with E. bo ule ng eri , b ut we fin d th at the tw o sp ecies 28 are all op atric an d repr ese nt two diverg ent clades (1. 77 % diver ge nt for 12 S– 16 S an d 5.39 % for 29 CY TB ). T hes e speci es can b e distin guish ed by the pr ese nce of a brig ht yello w blotc h in th e d orsal 30 ant erior regi o n of th e thig h in E. [to be rev eal ed] , w hich is abs ent or diffuse a nd pal e or cream in E. 31 bo ule ng eri . Epi p ed ob ates [to b e reve al ed] is the most nort her n species of E p ip edo ba t e s , whic h 32 exten ds sout hw ards al on g th e w estern ed ge of the An des. K no w n as the Ch ocó, thi s biog eo gra p hic 33 regio n h as b ee n lar gely conv erted t o a griculture i n Ecu ad or a nd is exp erie ncin g wid espre ad 34 transform atio n in C olo mbi a, w hich m ay en da ng er E. [to b e reve ale d] a n d bio diversity in the re gio n. 35 This version is a pr epri nt, an d w e o nly inte nd to na me t he sp ecies in th e final pu blish ed versio n. 36 37 Ke yw ord s: bio aco ustics, Chocó, m usic, me ga diverse, p oiso n frogs, n ew sp ecies, alp ha tax on omy, 38 DN A b arcodi ng. 39 40 Re su me n 41 El nú mer o d e es pecies d e a nfibi os descritas cad a a ño co ntin úa au me nta n do, es pec ialme nte en l as 42 regio n es tropical es, lo qu e im plica q ue la bio diversida d d e a nfibi os sigue sie n do su bestim ad a. 43 Describim os un a n u eva esp ecie de ra n a ven en osa de l as tierras baj as del P acífico del sur occide nte 44 de Col om bia: E pip e do bat es [to be r eve ale d] sp. nov., n om bra da así por el gé ner o d e música y d anz a 45 del P acífico con ocido co m o b am buco viej o o [to b e reve al ed] . L as ran as de esta es pecie ha bita n e n 46 bosq u es de tierr as baj as des de el nivel d e l mar h asta los 2 60 m. Este taxó n se difer encia de sus 47 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint cong é ner es por te n er un a com bi nació n d e ma nch as am arillas e n la re gió n d orsal a nterior d el m uslo y 48 los brazos, u na col oració n d orsal h om o gé ne a m arrón oscuro y cant os de adv ertenci a más lar gos y 49 en co nsecu e ncia con may or nú mer o d e p ulsos. Ta mbi én describim os la llam a da de cortejo d e E. [to 50 be rev eal ed], co n me n or frecue ncia pic o y duraci ón qu e la lla ma da de adv ertenci a. Los a nálisis 51 filoge n éticos confirma n la m o nofilia de la especi e y su posición co mo her ma n a d e Epi pe do b ates 52 narin e nsis, la cual se distribuye en el suro ccidente de Col om bia. E ntre l as especi es de 53 Epi pe do b ates, la n uev a esp ecie h a sid o p reviam ent e asig na d a a E. b o ule ng eri, per o las d os 54 especi es son al op átricas y represe nta n d os clados filo ge nética me nte diverg ent es (1.7 7 % div erg ent es 55 par a 12 S– 16S y 5.39 % p ara CY TB ). Esta s especies se p ue d en disting uir fen otípica me nte p or la 56 prese ncia d e u n a ma nch a a marilla brillant e e n la re gió n d orsal a nterior del m uslo e n E. [to be 57 reveal ed] , q u e está a use nte o difus a y pál ida o cre ma en E. b oul en geri. E pip ed o bat es [to be 58 reveal ed] es la esp ecie distribui da más al norte del gé ner o E pip ed o bat es , el cual se extien de hacia el 59 sur a lo larg o d el fla nco occid ent al de l a cordiller a d e los An d es. Esta regi ón co n ocida co mo el C hocó 60 bio ge ogr áfico, h a sido fu erte me nte tra nsforma d a p or agricult ura e n Ec ua d or y está experi me nta nd o 61 un a transfor mació n g e ner alizad a d e sus b osqu es e n C olo mbi a, lo cual po ne en peli gro a E. [to b e 62 reveal ed] y tod a su bi o diversida d e n u n fu turo cerca no. 63 64 I N TR OD UC T IO N 65 66 Epi pe do b ates sens u is a taxon of n eotro pi cal poiso n frogs (A nur a: D en dro bati da e) with ni ne put ative 67 species (Gra nt et al. (2 00 6, 2 01 7) a nd Ló pez-H ervas et al. (2 02 3): Epi pe d ob ates af f. espinos ai (see 68 Ló pez-H ervas et al. 2 0 23) , E. a nth o nyi (Nobl e 1 92 1), E. b oul en geri (B arb o ur 19 0 9), E. sp. 1 (see 69 Ló pez-H ervas et al. 2 0 23) , E. es pin osai (Fu nkh ous er 19 5 6), E. mach alilla (C olo ma 19 95), E. 70 macul atus (P eters 18 7 3) , E. nari ne nsis Mueses-Cis ner os et al. 2 00 8, an d E. tricolor (Boul en g er 71 18 99) . Fr ogs of th e g e nus Ep i ped ob a te s inh abit dry a n d h umid tro pical for ests from 0 to 1 80 0 m 72 across the P acific lowlan ds a nd f oot hills of the w estern sid e of th e A nd es of C olom b ia, Ecua d or, an d 73 north ern Per u (Gra nt et al., 2 00 6, 2 01 7). A rece nt assessme nt of g en etic an d p he n otypic diversity in 74 Epi pe do b ates fou n d several distinct evolu tionary lin ea ges wit h similar m orp hol ogy, i.e., cryptic 75 species (Ló p ez-Hervas et al. 20 23). It is t hus n o surprise th at pri or phylo g en etic studies, whic h 76 sampl ed differe nt, de e ply diverg ed cryptic species that w ere t ho ug ht to b elo n g to a single sp ecies at 77 the tim e, failed t o resolve phylo g en etic relations hips at th e sp ecies level (Clo u gh & Su mm ers, 20 00; 78 Grant et al., 20 1 7; Sa ntos et al., 20 14; T ar vin et al., 20 17; Ve nces et al., 2 00 3). 79 Previo us studies of Ep ip ed oba t e s bo u le nge r i fou nd hig h g en etic diversity and int erp op ulati on 80 variation i n aco ustic traits and larval m orp hol ogy, in dicatin g that Epi pe do b ates b oul en geri w as likely a 81 species com plex (L óp ez-H ervas et al., 20 23; L ötters et al., 2 00 3; S ant os et al., 20 0 9; Tarvin et al., 82 20 17). T h e most rece nt p hylo ge netic asse ssment (L óp ez-Hervas et al., 20 23) fo u nd that 83 Epi pe do b ates b oul en g eri sensu lat o cont a ine d repr ese ntatives of fo ur g en etically distinct linea ges: E. 84 bo ule ng eri (sensu stricto ), distribute d in sout hw estern Col om bia an d th e n orthw estern e d ge of 85 Ecua d or; E. espin osai, distrib ute d in ce ntral-wester n Ec ua dor; E. sp. 1, distribute d in n orthw estern 86 Ecua d or an d u p int o the s out her n e nd of the An de a n foot hills of Col om bia; a n d E. af f. espinos ai , 87 distribute d in ce ntral-w estern Ecua d or (Ló pez-H ervas et al., 2 02 3). In prior assessments of 88 ph en otypic diversity of this species, Lötter s et al (20 03) fo un d differ enc es in tem por al char acteristics 89 of the advertise me nt call bet we e n specim ens referr ed to as E. b oul en g eri from C olo mbi a (Anc hicayá, 90 Valle del Ca uca, corresp o ndi ng t o E. sp. 1 ) and E. b oul en geri fro m Ecu ad or (Lita, Im ba bur a, 91 correspo n din g to E. aff. espi nos ai ), while An ga n oy-Criollo an d C ep e da-Q uilin do ( 20 17) d escribe d th e 92 extern al mor ph olo gy of th e ta dp oles of E. bo ule ng eri from Col om bia (T um aco, N ariñ o, possibly 93 repres enti ng E. bo ule n geri sens u stricto ) an d from Ecua d or (Carchi, Esm eral d as, Imb ab ura, a n d 94 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint Sa nto Do min go provinces, id entificatio n u ncertai n, possibly corresp on din g to E. aff. espin osai , E. 95 bo ule ng eri sens u stricto , or E. espin osai ). Altho u gh th ey o nly exami ne d ta dp oles fro m the so uth ern 96 half of th e sp ecies distributio n ar ea of E. b oul en geri (i.e., excludi ng t he lin e ag e d escribed as E. sp. 1 97 by Ló pez-H ervas et al bio Rxiv), they foun d that th e Ec ua dori an po pul atio ns differe d from thos e of 98 Col om bia in bo dy sha p e, tips of mar gin al pa pilla e, an d p atter n of tail color atio n. Th u s, previous 99 evide nce poi nte d stron gly to E. bo ule n geri as a species com pl ex. 100 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint 101 Fig ure 1. M a p of re se arc h ar ea. T h e sou thern half of th e P acific lowla nds of Col om bia, p art of the 102 Ch ocó bi og e ogr ap hical re gio n, h osts at least three sp ecies of Ep i ped ob a te s . Ph oto gra ph of the fro g 103 from Tim biq uí (R eserva R a nita terribilis, F un daci ón Pr oAv es de Col om bia) w as o bta ine d from 104 iNatur alist (observatio n N o. 1 35 2 53 84 3 by Juan Carlos Lu na); ot her im ag es w ere ta ken by th e 105 aut hors. 106 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint 107 In this study, we d escribe a n ew sp ecies o f Epip ed o bat es from th e C olom bi an P acific lowla nds 108 correspo n din g to E. sp. 1 as assign ed by Ló pez-H ervas et al. (bi oRxiv). Using a co mbi natio n of 109 acoustic, mor ph olo gical, a nd m ol ecular a n alyses, we verify that this frog is a distinct an d pr eviously 110 unr ecog nize d speci es. We n am e th e n ew species E pip ed ob ates [to be rev eal ed] sp. nov. an d pr ovid e 111 a d etaile d d escription of its calls, morphol ogy, b eh avior, a nd nat ural history. As with many oth er 112 am phi bia n speci es, Epip e do bat es [to b e reveal e d] sp. nov. is un der thr eat from ha bi tat loss, pollutio n, 113 an d climate c ha ng e (Myers et al., 2 00 0; P ow ers & Jetz, 201 9; W arre n et al., 2 01 3). By ide ntifying a nd 114 describin g this n ew sp ecies, we ca n b ette r und erstan d th e diversity, evolutio n, g eo g rap hic 115 distributio n, ha bitat re q uirem ents, a nd co n servation ne eds of Epi pe do b ates speci es in Col om bia. W e 116 ho pe t hat o ur findi n gs will stimulate furt he r research a n d conserv ation efforts to prot ect the u niq u e 117 an d irrepl ace abl e a mp hibi an bio diversity of the C hocó bio ge o gra phical re gi on. 118 119 ME TH OD S 120 121 Ethic s st ate me nt 122 Scientific collecting proce dur es involvin g live ani mals follo we d pr otocols a p prove d b y the IAC UC at 123 the U niversity of Texas at Austin (A UC- 20 12- 00 03 2), the Univ ersity of California, Be rkeley (AUP-124 20 19- 08- 12 4 57), a nd C I CU AL of th e U niversida d d e los A nd es (PO E 1 8– 00 3). R esearch an d field 125 collection of sampl es w ere a uth orized by the Aut orida d N acio nal de Licencias A mbi ent ales (AN L A) of 126 Col om bia un der th e p ermiso marco r esolu ción N o. 11 7 7 to th e U niversida d d e los A nd es. Sp ecime ns 127 of E. b oul en geri fro m the typ e locality wer e collecte d u nd er R es. 06 1-2 01 6 by P arq ues N acio nal es 128 Nat urales d e C ol om bia. S am ples w ere exporte d u n der th e follo win g p ermits: CIT ES No. C O3 92 8 2, 129 C O4 14 43 , CO 46 948 , a nd ANL A No . 00 561 . 130 131 Sci entific c olle ctin g 132 We collecte d g e netic, mor ph olo gical, a nd acoustic dat a, as w ell as wh ole s pecim en s, of different 133 species a nd po pul atio ns of E pip ed ob ates in Col om bia ( Fig. 1; se e also Ló pez-H erva s et al. 20 23) 134 durin g 2 0 14, 2 0 16 an d 2 02 2. A nim als wer e e uth anize d by a n ov erd ose of lid ocai ne. Sex for most of 135 the sp ecime ns was det ermin ed at the tim e of collectio n b ase d o n w het her in dividu a ls were callin g or 136 not a n d w as confirme d by dir ect exami nati on of the go na ds after e uth a nasia. Bef ore fixation, liver an d 137 muscle sam ples w ere t ake n for mol ecul ar ge netic a nalyses a nd stor ed i n 9 5 % et ha n ol. In som e 138 cases, skin was remov ed f or qu a ntification of d efe nsive alkal oid co nte nt (see S u ppl . material 1). 139 Vo uch er specim ens w ere fixe d in 1 0 % ne utral b uffere d form alin an d transf erred directly to 70 % 140 eth an ol for lo ng-ter m storag e in t he am phi bia n collectio n of th e Mus eo de Historia N atural C.J. 141 Marink elle at th e U niversid ad de los A n de s (ANDE S:A) an d th e H erp etolo gy C ollection at the 142 Muse um of V ertebr ate Zo olo gy at th e Uni versity of California, B erkeley (M VZ: Her p) . As holotyp e a n d 143 par atypes w e d esig nat e individ u als that w ere collecte d in Au gust 2 0 22 fro m L adrille ros, 144 Bu en ave ntur a, Vall e d e C auc a, Col om bia (see T abl e 1). 145 146 Morp h olo gy 147 Foll owi ng previo us descriptio ns of n ew Ep ipe do bat es species (Cisn eros-H ere dia & Yá nez-M uñ oz, 148 20 10; M u eses-Cisn eros et al., 2 00 8), w e took 1 3 stan d ardize d m orp ho metric me asu reme nts after 149 fixation (Watt ers et al., 20 16): S n out-ve nt-len gth (S VL); for earm l en gth ( FA L), taken from tip of th e 150 flexed elb ow an d th e proxim al e dg e of th e palm ar tu bercle; h an d le n gth (H aL), m ea sured fro m th e 151 proxim al ed g e of th e larg e m edi al p alm ar tub ercle to th e tip of th e lo ng est (third) fing er; tibia le ngt h 152 (TL), take n from th e o uter surfac e of th e flexed k ne e to th e h eel infl ection; fo ot le ngt h (FL), take n fro m 153 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint the pr oximal ed ge of the extern al m etatar sal tub ercle to the en d of th e fo urth to e; h ea d wi dth (H W), 154 taken bet we en t he an gles of th e ja ws; he a d len gth ( HL), dista nce from t he tip of the snout to t he 155 an gle of the j aw; dista nce fro m the ce nter of on e n ares to th e a nteri or e dg e of th e ey e (NE D); eye 156 width ( EW); eye- nar es distanc e (EN D); intern arial dista nce (IND); int erocul ar dista nce (IOD); a nd 157 horizo ntal di am eter of the tymp a nu m (T D). All meas urem e nts were t ake n u nd er a di ssecting 158 microscop e usin g a di gital calip er at a r esolutio n of 0.0 1 mm. W e co nd ucted a Kr uskal-Wallis test to 159 comp are bo dy size (snout-ve nt-len gth) across species an d a post-h oc pair ed co mp arison using a 160 Wilcoxon ra nk-sum test. W e also co nd ucted an AN OV A to assess sexual dim orp hism in b ody size, 161 previo usly checked its assum ptions. 162 163 We als o review e d a nd su mm arized sp eci es-level p he noty pic variation as describe d in Ló pez-H ervas 164 et al. (20 23) to provid e a sp ecies-level assessment of col or p attern vari ation t hat ca n b e use d to 165 disting uish species. 166 167 M o l ecu l a r an d p hy l oge ne t i c ana l ys es 168 DN A w as extracted fro m liver an d m uscle tissue using t he Qi ag e n D Ne asy Bloo d & Tissue kit 169 (Vale ncia, C A) follo win g th e ma n ufactur er' s protocol. W e seq ue nce d tw o mitoch o ndr ial ge n e 170 fragm ents from 5 in dividu als of E pip ed ob a tes [to be rev eal ed] sp. n ov. collected fro m the typ e 171 locality: a fragm ent incl udi ng parts of th e 12 S a nd 16 S mitoc ho ndri al rRN A g en es (69 2 b p) inclu din g 172 the int erveni ng vali ne t RN A g en e ( 12 S– 16S ), an d cytochrom e b ( CY TB ; 6 5 9 b p). W e use d th e sam e 173 primers a nd prot ocols as describ ed i n L óp ez-Hervas et al. (bioRxiv). Briefly, the P C R prot ocols 174 follow ed Go e bel et al., (199 9), S ant os an d Ca nn atella ( 20 11), an d S ant os et al. (20 0 3). PC R 175 pro ducts wer e seq u ence d in bot h directio ns at the Fu nction al G en omics L ab orat ory at the U niversity 176 of Califor nia, B erkel ey. Th e chro mat ogr a ms were trim me d a nd c onse nsus co ntigs wer e o btain e d 177 using sa n ger an alyseR (C ha o et al., 20 21) in R v.4.3.1 (R Cor e T ea m, 2 02 3). Ne w seq ue nces w ere 178 de posite d in G e nB ank (accessio n n um ber s OR7 89 87 5 –O R7 8 98 84). 179 180 We ali gn e d the five n e w seq ue nces usi ng MU SC LE v3. 8.3 1 (Ed g ar, 20 0 4) in AliVi e w (Larsso n, 20 1 4) 181 with existing 12 S– 1 6S an d CY TB d ata for 10 8 ot her Ep i ped ob a te s individ u als, includin g o ne 182 out gro up ( Silversto nei a n ubic ola ) a nd 4 in dividu als assign ed to E p ipe do ba t e s sp. 1 by Ló pez-H ervas 183 et al. (20 23), w hich corres po nd h ere to E p ipe do bat es [to b e reve ale d] sp. nov. Usin g the resultin g 184 alig nm ent (D ata S1), w e calculat ed me an pairwis e p-dista nces (u ncorrecte d) b etw e en sp ecies for 185 each ge n e with th e R script provi de d by L óp ez-Hervas et al. (20 23) with a pe v 5.7.1 (Para dis & 186 Schlie p, 2 01 9), using pair wise d eleti on for missing d ata. F ollo win g L óp ez-Herv as et al. (20 23), w e 187 exclud ed th e in dividu als from th e E. aff. e spinos ai Bilsa a nd La Tort ug a localities, w hich sho w 188 evide nce of introgr ession with E. m ach alilla . 189 190 We o btai ne d a d dition al se qu enc es of thre e n uclear m arkers (total le ngt h of 2 22 7 b p ), nam ely histon e 191 H3 ( H3 ), b on e m orp ho ge n etic protei n 2 ( BM P2 ), an d volta ge- gat ed pot assium ch an nel 1. 3 ( K V 1. 3 ), 192 as well as th e mitoc ho ndri al control re gi on ( CR; 1 03 1 b p) from pr evio usly publish e d sequ e nces of th e 193 same 10 8 in dividu als refer ence d a b ove (S up pl. mat erial 1). A m axim um likelih oo d p hylog e ny was 194 estimate d usi ng this ex pa nd e d alig nm e nt in IQ-T RE E v2.0. 3 (Kalya a na mo orthy et al ., 201 7; Min h et 195 al., 20 20) a n d p ara met ers describe d in L ó pez-H ervas et al. (bio Rxiv). Briefly, we p artitione d pr otein-196 codin g g e nes by g en e a n d by cod o n p osition. W e exclu de d th e first and seco nd c od on positio ns of 197 BM P a nd H3, w hich w ere i nvaria nt across all sampl es. We sel ected th e m od el of evolutio n a n d d ata 198 partitio ns un der th e B ayesia n Inf ormati on Criterion usin g o ption - m T ES TM ER GE. T o assess bra nch 199 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint supp ort, we perf orm ed 10, 00 0 ultraf ast bo otstrap re plicates (Min h et al., 2 0 13) a n d plotte d th em ont o 200 the o ptim al likeliho o d tree in Su p pl. materi al S2. 201 202 Call re cordi ng an d bio ac ou stic an aly se s 203 We recor de d th e a dvertisem e nt calls of twelve m ale fro gs of Epi p ed ob ates [to b e re veale d] sp. n ov. 204 from thre e V alle de Ca uca localities un der natur al con ditio ns: Anchicay á (four in dividu als), Pian g uita 205 (six), and fro m the typ e locality, La drillero s (two). We also rec ord ed five m ales of E. bo ule ng eri from 206 the type l ocality (Isla Gorg on a, C auca, Co lom bia), five mal es of E. b oul en g eri from Mara grícola 207 (Tum aco, N ariñ o, C olo mbi a) a nd six male s of E. narin ensis from its type locality (Bioto po Nat ural 208 Reserve, Nari ño, C olo mbi a). All recordi ng s were d e posite d in F o nozo o (F Z-S OU ND -COD E 1 4 65 7-209 14 68 5). R ecordi ngs w ere t aken duri ng th e morni ng ( 7– 13 h) usin g a u ni direction al m icroph on e 210 (Se nn heiser K6/ ME 66) co nn ecte d to a dig ital record er (Mara ntz PM D 66 0/Z oo m H 4n Pro) a nd 211 positio ne d 5 0– 1 50 cm in fro nt of a calli ng mal e. Imme di ately after recor din g, w e me asure d th e 212 temp erat ure of the su bstrate using an i nfrare d ther mo met er (Oakto n m od el 3 56 2 9). We di gitized th e 213 recordi ngs at a minim u m of 1 6-bit resol ution an d 4 4.1 kHz sam plin g rat e in R AV EN PR O 1.6 214 (Bioac oustics Rese arch Pr ogr am, 2 01 4). For th e termi nol ogy a n d proc ed ures for m easuri ng call traits 215 we foll ow Kö hler et al. (20 17). T h e spectrogr ams a nd oscillogra ms wer e gr ap he d u sing S ee wav e R 216 packa ge (S u eur et al., 20 08) with a n F FT win do w usin g th e Bl ackman alg orithm, wi nd ow l en gth of 217 25 6 sam ples a n d overl ap of 90 % . 218 219 We d efin ed a call as th e sou nd pro duc ed durin g a sin gle ab d omi nal m uscular co ntraction (Er dtm an n 220 & Am ézq uita, 2 00 9; K öhl er et al., 2 01 7). In E pip ed ob ates , such co ntractio ns pro duc e a sin gle " b uzz" 221 comp ose d of sever al p ulses (Brow n et al., 20 11; Er dtm an n & Am ézquit a, 2 00 9; Mye rs & Daly, 19 76). 222 We a n alyzed five cons ecutive calls per in dividu al by m easuri ng t heir p eak fre qu enc y (the frequ e ncy 223 with th e hig h est am plitud e), low fre q ue ncy (the low er freq u ency of th e call), high fre q ue ncy (the u pp er 224 frequ e ncy of the call) a nd 90 % b an dwi dth (-10 d B thres hol d; cont ainin g th e freq u enc y rang e that 225 enco m passes 9 0 % of the so un d e n ergy in each call). Hig h a n d low fre q ue ncies of th e calls were 226 me asure d at 20 dB (re 20 m PA) bel ow th e pe ak inte nsity of the p eak freq u ency. At this value th e 227 signal en ergy co uld still be clearly disting u ished fro m th e b ackgro un d n oise. We also meas ure d th e 228 followi ng t em por al variabl es: call dur ation, num b er of p ulses per call a n d inter-call in terval. For E. [to 229 be rev eal ed] sp. n ov., we also m easur ed t he pulse r ate, p ulse d urati on, a n d inter-p u lse interval. Call 230 par am eters for the calls of e ach i ndivid ual were avera g ed to statistically comp are a mo ng i ndivid uals. 231 To avoid r ed un d ancy am o ng th e ac oustic variabl es, we us ed a Princip al C om po n ent An alysis (PCA) 232 to red uce th e n um b er of p aram eters im ple me nte d in th e ‘PC A’ fu nction of the ‘ Facto Min eR’ R 233 packa ge ( Lê et al., 20 08). In ord er to ide nt ify wheth er E. [to b e reve ale d] sp. n ov. could b e 234 differe ntiate d b ase d o n a dvertisem ent call s, we ran a L DA on th e first 5 PCs usin g the l da' functio n of 235 the M AS S R packa ge ( Ve na bles & Ripl ey, 20 02). W e utilized an Kruskal-W allis test to exa min e the 236 releva nt call traits (peak freq u ency, call du ration an d n um b er of p ulses per call) th at differe ntiate E. 237 [to be rev eal ed] sp. n ov. from ot her sp ecie s and a p ost-hoc p aire d com pariso n usin g a Wilcoxon 238 rank-sum test. 239 240 Re po sitorie s, In stitution al a cro ny ms, a nd In stitutio nal ab bre viati ons 241 Thr ou gh o ut our pa per we use gl ob ally uni qu e id entifiers (GUI Ds, Glob ally Uni qu e Id entifiers T ask 242 Grou p, 2 01 1) to ref er to th e vouch er sp ecime ns at QC A Z (QC AZ: A:X XX X, Mus eo d e Z ool ogí a d e la 243 Po ntificia Universid ad Cat ólica d el Ecu ad or, Quito, Ec ua d or), the M VZ (M VZ: Her p: XX XX, 244 her pet olo gical collectio ns of the M use um of Vert ebr ate Z o olo gy, University of Califo rnia, B erkeley, 245 US A), an d th e a mp hibi an coll ection at the Muse o d e Historia Nat ural C.J. M arinkelle at the 246 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint Universid ad de l os An des, B og otá, Col om bia (A ND ES: A:X XX X), in a n effort to facilitate fut ure 247 machi ne-r ea da bility of this text. 248 249 RE SU LT S 250 251 Sy ste matic s 252 Epi pe do b ates [to b e reve ale d] sp. nov. 253 Pro pose d E n glish com mo n n am e: [to b e reveal e d] Nurse Fro g. 254 Pro pose d S p anish co mm on na m e: Ra n a no driza d e [to b e reve ale d]. 255 256 257 Fig ure 2. Im ag e s in life a nd in pre ser va tive of th e h olot yp e of E pip e do bat es [t o b e rev e ale d] 258 sp. n o v. . A F ull specim en in life; B Dors al view in life; C V entr al view in life; D Lat eral view in life; E 259 Dorsal vie w in pr eservative (7 0 % et h an ol); F Ve ntral view i n pres ervative; G L ateral view in 260 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint preservative; H Dorsal ha n d in pr eservative; I Ve ntral h an d in pr eservative; J Dorsal foot in 261 preservative; K Ve ntral fo ot in pr eservative. Scale bars re prese nt 5 m m (B to G) or 2.5 m m (H to K). 262 263 Ta ble 1. Mor ph om etric ch ara cter s an d su mm ary statisti cs of t he m ale s o f typ e serie s of 264 Epi pe do b ate s [to b e re ve ale d] sp. n ov. Me asur em ents (in m m) of a dult fe m ales an d mal es; valu es 265 are giv en as me an, S D, a nd r an ge. Se e M eth ods section f or expl an atio n of a bbr evia tions. *Holoty pe. 266 267 Mus eum Cod e Cole ctor Cod e Se x SVL FAL HaL TL FL HW HL NED EW END IND IOD TD A N DE S -A 5 254 M B C 753 Male 17 .2 4. 3 3. 7 7. 7 7 5. 4 6. 3 2. 5 2. 1 1. 6 2. 1 2. 9 0. 9 A N DE S -A 5 256 A J C0 773 1 Male 1 7. 20 4. 10 3. 90 7. 80 6. 90 5. 80 6. 90 2. 90 2. 10 1. 60 2. 20 3. 30 1. 00 A N DE S -A 5 260 A J C0 773 5 Male 1 7. 40 4. 40 3. 70 7. 90 7. 00 5. 60 6. 40 3. 00 2. 50 1. 60 2. 50 3. 80 1. 00 A N DE S -A 5 261 A J C0 773 6 Male 1 8. 70 4. 50 4. 30 8. 50 7. 30 5. 90 7. 00 3. 30 2. 70 1. 70 2. 50 3. 80 1. 00 A N DE S -A 5 263 A J C0 773 8 Male 1 7. 50 4. 60 3. 80 7. 70 6. 60 6. 00 7. 30 3. 00 2. 40 1. 60 2. 30 3. 50 0. 90 M V Z : He r p: 295 434 A J C0 774 2 Male 1 6. 70 4. 00 3. 60 7. 50 6. 70 4. 90 6. 70 2. 80 1. 90 1. 70 2. 30 3. 20 0. 90 M V Z : He r p: 295 436 A J C0 774 4 Male 1 6. 70 3. 80 3. 70 7. 10 6. 70 5. 70 6. 90 2. 70 2. 40 1. 70 2. 10 3. 40 1. 00 A N DE S -A 5 259 R D T 09 35 Male 1 6. 90 4. 40 3. 80 7. 90 7. 20 5. 30 6. 60 3. 60 2. 40 1. 80 2. 30 3. 80 0. 90 Mea n 1 7. 29 4. 26 3. 81 7. 76 6. 93 5. 58 6. 76 2. 98 2. 31 1. 66 2. 29 3. 46 0. 95 SD 0. 64 0. 27 0. 22 0. 40 0. 25 0. 36 0. 33 0. 35 0. 26 0. 07 0. 16 0. 33 0. 05 Min 1 6. 70 3. 80 3. 60 7. 10 6. 60 4. 90 6. 30 2. 50 1. 90 1. 60 2. 00 2. 90 0. 90 Max 1 8. 70 4. 60 4. 50 8. 70 7. 60 6. 20 7. 80 3. 60 2. 70 2. 00 2. 50 4. 20 1. 20 268 Ta ble 2. M orp ho metric c har act ers an d s um mar y st atistic s of t he f em ale s of typ e serie s of 269 Epi pe do b ate s [to b e re ve ale d] sp. n ov. Me asur em ents (in m m) of a dult fe m ales an d mal es; valu es 270 are giv en as me an, S D, a nd r an ge. Se e M eth ods section f or expl an atio n of a bbr evia tions. *Holoty pe. 271 272 Mus eum Cod e Cole ctor Code Se x SVL FAL HaL TL FL HW HL NED EW END IND IOD TD AND ES- A 52 5 5* M B C 754 Fem ale 1 8. 50 4. 60 3. 90 8. 10 6. 90 5. 70 6. 80 3. 00 2. 30 1. 70 2. 30 3. 70 0. 90 A N DE S -A 5 257 A J C0 773 3 Fem ale 1 9. 50 4. 50 4. 50 8. 70 7. 20 6. 00 6. 70 3. 20 2. 60 2. 00 2. 40 4. 20 1. 10 A N DE S -A 5 262 A J C0 773 7 Fem ale 1 8. 30 4. 40 3. 90 7. 70 6. 70 6. 10 7. 00 3. 20 2. 40 1. 80 2. 00 3. 60 1. 10 A N DE S -A 5 264 A J C0 773 9 Fem ale 1 7. 50 4. 40 3. 90 8. 20 7. 20 5. 70 7. 00 2. 90 2. 40 1. 90 2. 40 3. 60 0. 90 M V Z : He r p: 295 433 A J C0 774 0 Fem ale 1 9. 10 4. 30 4. 00 8. 00 7. 20 6. 20 7. 10 3. 10 2. 30 1. 90 2. 40 3. 40 1. 20 M V Z : He r p: 295 435 A J C0 774 3 Fem ale 1 8. 90 4. 60 4. 20 8. 60 7. 60 5. 80 7. 10 2. 90 2. 20 1. 70 2. 10 3. 40 1. 10 M V Z : He r p: 295 432 R D T 09 34 Fem ale 1 9. 10 4. 30 4. 00 8. 00 7. 00 5. 90 7. 80 2. 90 2. 40 1. 60 2. 40 3. 50 0. 90 A N DE S -A 5 258 A J C0 773 4 NA 1 8. 60 4. 40 3. 60 7. 30 6. 60 6. 00 6. 50 3. 40 2. 30 1. 90 2. 30 3. 90 1. 00 Mea n 1 8. 70 4. 44 4. 06 8. 19 7. 11 5. 91 7. 07 3. 03 2. 37 1. 80 2. 29 3. 63 1. 03 SD 0. 66 0. 13 0. 22 0. 35 0. 29 0. 20 0. 35 0. 14 0. 13 0. 14 0. 17 0. 28 0. 13 Min 1 7. 50 4. 30 3. 90 7. 70 6. 70 5. 70 6. 70 2. 90 2. 20 1. 60 2. 00 3. 40 0. 90 Max 1 9. 50 4. 60 4. 50 8. 70 7. 60 6. 20 7. 80 3. 20 2. 60 2. 00 2. 40 4. 20 1. 20 273 Materi al ex ami ne d 274 275 Hol oty pe 276 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint CO LO MBIA • ♀; L adriller os, Bu en ave ntu ra, Valle del Ca uca; 3. 94 5 22 1, −7 7.3 64 99 3; 28 m. a.s.l.; 6 277 Au g. 2 02 2; collectors Re b ecca D. T arvin, Mileidy B eta nco urth-C un d ar, Jua n C amilo Ríos-Orju ela; 278 AN DE S:A:5 2 55. 279 280 Parat y pe s 281 CO LO MBIA • 4 ♀♀, 6 ♂♂, 1 ND; sa me dat a as for h olotyp e; A ND ES:A: 5 25 4, 5 25 6– 52 6 5 • 3 ♀♀, 2 282 ♂♂; same d ata as for hol otyp e; Ge nb a nk: OR7 89 8 80 –8 4 a n d OR 7 89 87 5 –7 9; MV Z :Herp: 29 5 43 2–283 29 54 3 6. 284 285 286 287 Other m ateri al 288 289 CO LO MBIA 290 • Epip ed o bat es [to be r eveal ed] 2 ♀♀, 6 ND; Ty pe loc ality, Ladriller os, Bu en ave ntu ra, Valle del 291 Ca uca; 3. 95 08 8 2, −7 7.3 58 29 3; 5 3 m a.s.l.; 26 N ov. 20 14; coll ectors Re becca D. Tar vin an d Fr ay 292 Arriag a; Ge n Ba nk: OR 48 9 01 1– 1 2, OR 17 97 90 – 91, O R7 3 47 03 – 04, O R1 7 98 35 – 36, OR 17 98 7 5, 293 OR 17 98 8 0; AN DE S:A: 24 5 8– 24 6 5. Th e skin was re mov ed prior to pr eservatio n for e valuati on of 294 alkaloi d cont ent fro m thes e in dividu als, with the excepti on of AN DE S:A: 24 64. • E. [to be reve al ed] 1 295 ♀; La B arra, B ue n avent ura, V alle del C a u ca; 3.98 5 06 4, -77. 37 6 72 3; 1 5 m a.s.l.; 26 Nov. 2 01 4; 296 collectors Re becca D. Tarvin an d Fr ay Arriag a; Ge nB a nk: OR 48 90 1 0, OR 17 9 78 9, OR 73 47 0 2, 297 O R1 79 834 , O R1 798 51 ; AN DE S :A : 24 55 . • E. [to be rev eal ed] 5 ♂♂; Pi a ng uita, B ue nave ntur a, V alle 298 del C a uca; 3.8 4 19 54, -7 7. 19 87 1 8; 17 m.a .s.l.; 12 Sep. 2 0 16; collect ors Re becca D. Tarvin, Mil eidy 299 Bet anco urth- Cu nd ar, S an dra V. Flech as; AN DE S:A:3 6 90- 94. • E. [to b e reve ale d] 4 ♀♀, 3 ♂♂, 300 Da nu bio, Da gu a, V alle d el C auc a; 3.6 1 15 28, -7 6.8 85 1 94; 7 0 5 m a.s.l.; 5 Nov. 2 01 6; collectors 301 Mileidy B eta nco urth-C un d ar, Ad olfo A m ézquita, an d Ivan Beltrá n; G en Ba nk: OR 48 9 01 3, OR 1 79 79 2, 302 O R7 34 705 , O R1 798 37 ; AN DE S :A : 37 14 –2 0 . 303 304 • E. bo ule ng eri 3 ♀♀, 4 ♂♂, 1 N D; Isla G org on a, Gu a pi, Ca uca; 2. 96 4 65, -7 8.1 73 6 85; 2 0 m a.s.l.; 305 12 Se p. 2 01 6; collectors Re b ecca D. T arvin, Mileidy B eta nco urth-C u nd ar a nd Sa ndr a V. Fl echas; 306 Ge nB ank: O R O R4 88 9 92, O R1 7 97 71, O R7 34 6 81, O R1 7 98 12; AN DE S:A:3 6 95- 37 02. • 1 N D; Isla 307 Gorg o na, G u api, C auc a; 08 Se p. 2 00 5; A ND ES: A:0 56 0; C ollector u nkn ow n . • E. b oul en geri 3 ♀♀, 4 308 ♂♂, 1 ND; Isla G org on a, Gu a pi, Ca uca; 2.9 64 65, -7 8.1 7 36 85; 20 m a.s.l.; 12 S ep. 20 16; coll ectors 309 Re becca D. Tarvin, Mil eidy B eta nco urth-C un dar an d S an dra V. Flec has; G en Ba nk: OR O R4 8 89 92, 310 O R1 79 771 , O R7 346 81 , O R17 98 12 ; AN DE S : A :3 695 - 37 02 . • E. bo ul en geri 8 ♀♀; Mara grícola, 311 Tu mac o, Nari ño; 1. 68 0 84, -7 8.7 49 2 4; 7 m a.s.l.; 09 Dec. 2 01 4; collectors Re b ecca D. Tarvin, Mil eidy 312 Bet anco urth- Cu nd ar, Cristian Flórez; G en Ba nk: OR 48 89 9 0, OR 17 9 76 9, OR 7 34 67 9 , OR1 79 8 10, 313 O R1 79 862 , O R4 889 91 , O R17 97 70 , OR 73 46 80 , OR 17 981 1 , OR 179 86 3 ; A ND ES : A: 2 46 8– 75 . • E. 314 bo ule ng eri 1 N D; L a N utria, El Diviso, B arbaco as, Nari ño; 1.3 60 83, -7 8.1 8 07 6; 7 62 m a.s.l.; 10 D ec. 315 20 14; coll ectors Re becca D. Tarvin, Mil ei dy Bet anco urth- Cu nd ar, Cristian Flórez; G en Ba nk: 316 O R4 88 977 , O R1 797 56 , O R73 46 63 , OR 17 97 94 , OR 17 984 0 ; AN DES : A : 247 6 . 317 318 • E. narin ensis 3 ♀♀, 5 ♂♂, 1 N D; R eser va Nat ural Bi oto po, B erlín, B arb aco as, Na riño; 1.4 0 89 99, -319 78. 28 12 4 6; 51 8 m a.s.l.; 25 Se p. 2 01 6; co llectors Reb ecca D. T arvin, Milei dy Bet an courth-C u nd ar 320 an d Cristian Flór ez; Ge nB ank: O R4 89 0 08 , OR1 79 7 87, O R7 3 47 00, OR 17 98 3 2, OR 48 90 0 9, 321 O R1 79 788 , O R7 347 01 , O R17 98 33 ; AN DE S : A :3 703 –3 711 . • E. nari ne nsis 10 ♂♂; Reserva Nat ural 322 Bioto p o, Berlín, Bar baco as, Nari ño; 1.4 11 26 3, -78. 28 5 09 9; 6 00 m a.s.l.; 22 Jul. 20 0 6; collectors 323 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint Vivian a M ore no-Q ui ntero, Jo nh Jair o M ue ses-Cisneros, L uisa M erce des Br avo, C arol Narvá ez, a nd 324 Bie nveni d o Cort és; ICN 5 33 4 4 (hol otyp e), ICN:5 33 3 6– 53 3 40, 5 3 34 2– 5 33 46 ( par atypes). 325 • Andi no b ates min utus 4N D; La Barr a, Bu en ave ntura, Vall e d el Ca uca; 3. 98 5 06 4, -77. 37 67 2 3; 15 m 326 a.s.l.; 25 Nov. 2 01 4; collectors R eb ecca D . Tarvin a nd Fray Arria ga; AN DE S:A 24 51 –5 4. • A. min utus 327 1N D; L adriller os, Bu en ave ntura, Valle del Ca uca; 3. 94 52 2 1, −7 7.3 64 99 3; 2 8 m a.s.l; 6 Au g. 2 02 2; 328 collectors Re becca D. Tarvin, Mil eidy B et anco urth- Cu nd ar, Jua n C amil o Ríos-Orj u ela; 329 AN DE S:A:5 2 66. 330 331 EC UA DO R 332 • Epip ed o bat es aff. espin osai 1 ND; Lita, C archi; 12 Au g. 1 99 2; collector B ue n o M.; ICN: 32 50 4. 333 334 D i ag no s is 335 Epi pe do b ates [to b e reve ale d] is a small d en dro bati d frog (S V L me a n = 1 7.9 9 mm a nd SD = 0. 95 336 mm, N = 1 6 fro gs; Ta ble 2) characterize d by havin g a unif ormly bro wn dorsal col ora tion exte n din g 337 throu g ho ut the lim bs, an d a brig ht yellow blotch in t he co nce ale d re gio n of th e groi n . For a d etail ed 338 comp arison of the c olorati on of E. [to b e reveal e d] sp. nov. with oth er species of th e ge nus se e sup pl. 339 materi al S 3. Th e first finger is lon ger th an the seco n d a nd th e third fin g er is swollen i n mal es. In o ur 340 revision of th e m orp hol ogy, w e n ote d that fingers II an d IV ar e re duc ed in l en gth i n E . [to be reve ale d] 341 (Fing er II/SV L: me a n = 1 6.1, S D = 1.5 9 %; Fing er IV/S V L: me an = 15. 1, SD = 1.4 2 % , N = 37 fro gs) 342 (Fig. 2 H–I) a nd E. n arine nsis (Fin ger IV/S VL: m ea n = 1 6.9, S D = 0. 85 %; Fin g er IV/SV L: me a n = 16. 2, 343 SD = 1. 08 % , N= 8 frogs) relative to E. b oul en geri - Go r gon a (Fi ng er IV/S VL: me an = 1 9.2 %, SD = 344 0.6 7 %; Fi ng er IV/S VL: m e an = 1 8.2, SD = 1.12 % , N= 9 frogs), E. b oul en g eri -Nariñ o ( Fin ger IV/ SV L: 345 me an = 17. 5, SD = 0.9 9 %; Fi ng er IV/S VL: mea n = 17. 7, SD = 2.1 4 %, N= 9 frogs) or E. aff. espin osai 346 (Ta ble 3, su p pl. materi al S 3). Besid es its coloratio n, E. [to b e reve ale d] sp. n ov. differs in the 347 structure an d com p osition of its adv ertiseme nt calls, with lon ger calls a nd i n turn a gre ater n um ber of 348 pulses th an oth er E pip ed ob ates sp ecies, but in seri es of only o n e call. A sum mary of the 349 morp h olo gical char acters of E. [to b e reve ale d] sp. nov. com p are d to ot her Ep i ped ob a te s species in 350 Col om bia is sho wn i n T abl e 3. W e d escribe b elo w in d etail th e a dvertisem ent calls a nd m orp h olo gical 351 traits of E. [to be reve ale d] sp. n ov. in com pariso n with its con ge n ers, and an assessme nt of its 352 ge netic diver ge nce fro m oth er speci es in this ge nus. 353 354 T ab l e 3 . C ha r ac t e r i s t ic s o f m o rp ho l ogy, c o l o ra t i o n , a nd ca l l s o f E. [to be r ev ea led] sp . no v . 355 co mp are d to ot her sp eci es of E pip ed o bat es . F or com p arison of mor ph olo gical structures, we us ed 356 specime ns d ep osited at the I nstituto d e Ci encias N atur ales of th e U niversida d N acio nal d e C olo m bia. 357 For E. narin e nsis w e r e v i ew ed t he ho l o t y pe ( I C N :5 33 44 ) an d t y pe s e r i e s ( I C N :5 3336 –5 334 0 , 533 42 –358 53 34 6). F or E. aff. espi nosai, w e revie we d only o ne sp ecime n pr ese nt in this collection (IC N:3 25 0 4) 359 an d th e b ody size dat a w ere provid ed by Ló pez-H ervas et al. (2 02 3). T he ta bl e is present ed i n p arts 360 across the n ext several p ag es for re ad abil ity. 361 362 S p ec ies Snout- Vent Length (mm) (mea n ± SD) Fing er Lengt h I> II Fing ers II and IV redu c e d Fing er III sw el lin g in adult m al es Webbi ng : Toe II P o s tax ia l Webbi ng : Toe II I Pr ea xi al Webbi ng : Toe II I P o s tax ia l Webbi ng : Toe IV Pr ea xi al Webbi ng: Toe IV P o s tax ia l .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint E. [t o be reve al ed ] 16 .9 2 ± 0. 47 , N = 9 Ye s Ye s Ye s Ab se nt Ab se nt Pres e nt Pres e nt Pres e nt, ext e nds be yo n d t h e ba sal su bartic ul a r tu b ercle o n to e IV E. nari ne nsis 19 .9 2 ± 0. 47 , n= 8 Ye s Ye s Ye s Pres e nt, r e du ce d Ab se nt Pres e nt Pres e nt Pres e nt, ext e nds t o or ju st bel ow th e ba sal su bartic ul a r tu b ercle o n to e IV E. bo ul en g eri - Gor g on a 17 .6 9 ± 0. 11 , n= 39 Ye s No Ye s Ab se nt Ab se nt Pres e nt Pres e nt Pres e nt, ext e nds be yo n d t h e ba sal su bartic ul a r tu b ercle o n to e IV E. bo ul en g eri - Nariñ o 17 .3 7 ± 0. 67 , n= 9 Ye s No Ye s Ab se nt Ab se nt Pres e nt Pres e nt Pres e nt, ext e nds be yo n d t h e ba sal su bartic ul a r tu b ercle o n to e IV E. af f. es pin os ai 17 .1 1 ± 1. 69 , n= 28 Ye s No Ye s Ab se nt Ab se nt Ab se nt Ab se nt Pres e nt, ext e nds be yo n d t h e ba sal su bartic ul a r tu b ercle o n to e IV 363 S p ec ies Rel ati ve Fing er Length s Rel ati ve Toe Length s Metat ar sa l Fol d Dors al sk in textur e Dora l colo r Obli que Late ra l Strip e colo r Obli que Late ra l Strip e morpho log y Vent rol ate ra l str ip e E. [t o be reve al ed ] III> I>I I> I V IV> III >V >II > I Ab se nt Finely g r anu l a te d Dark brow n, red dis h brow n Pres e nt, yellow po st eriorl y and co pp er an teri orly Com plet e Ab se nt .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint E. nari ne nsis III> I>I I> I V IV> III >V >II > I Ab se nt Finely g r anu l a te d Dark g r een Pres e nt, light g r een Only r e a che s up to 1/ 4 of t he bo dy le ng th from th e groi n Pres e nt, lig ht g r een E. bo ul en g eri - Gor g on a III> I>I I> I V IV> III >V >II > I Ab se nt Finely g r anu l a te d Brow n, red dis h brow n Pres e nt, cream , narr ows an teri orly Com plet e Pres e nt, whit e E. bo ul en g eri - Nariñ o III> I>I I> I V IV> III >V >II > I Ab se nt Finely g r anu l a te d Red dis h brow n with dark sp ots Pres e nt, cream po st eriorl y and co pp er tow ard s th e an teri or regi on Com plet e Pres e nt E. af f. es pin os ai III> I>I I> I V IV> III >V >II > I Ab se nt Finely g r anu l a te d Dark brow n, red dis h brow n Pres e nt, cream po st eriorl y and co pp er tow ard s th e an teri or regi on Com plet e Pres e nt 364 S p ec ies Abdom en colo rati on Abdom en patter n Late ra l sh ape of snout Dors al sh ape of snout Tym pan um/ E y e (%) HW/S V L (%) HW/H L (%) Spot on ante riodo rs al sid e of thigh E. [t o be reve al ed ] T u r quo i s e blu e wi th blac k sp ots Hea vily mottl e d Proj ecti ng , p r ot r ud i ng Slig htly r o und e d 3 3. 3–6 6. 0 27 .7 – 36 .3 68 .4 – 10 3. 7 Pres e nt, co nspi cu o us, an d i nt e nse yellow E. nari ne nsis Lig ht oliv e g r een w i th blac k sp ots Sp eckl e d or slig htl y mottl e d Proj ecti ng Slig htly r o und e d 5 3. 8–5 8. 9 29 .8 – 33 .7 79 .3 – 87 .6 Ab se nt , yellow , or diff us e E. bo ul en g eri - Gor g on a Cream y whit e wit h blac k sp ots Mottl e d Proj ecti ng , p r ot r ud i ng Slig htly r o und e d 4 4. 8–5 9. 4 28 .7 – 33 .2 80 .4 – 10 3. 2 Pres e nt, cream , an d ext e nd e d alo n g t h e thi gh E. bo ul en g eri - Nariñ o Bluis h whit e wit h blac k sp ots Mottl e d or with i s o l at ed sp ots Proj ecti ng , p r ot r ud i ng Slig htly r o und e d 4 5. 0–6 4. 0 25 .7 – 32 .2 76 .4 – 86 .6 Ab se nt .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint E. af f. es pin os ai Whit e or cream wit h larg e blac k sp ots Mottl e d or with l arg e sp ots Proj ecti ng , p r ot r ud i ng Slig htly r o und e d NA NA NA Ab se nt or diff us e cr ea m- c o l o red 365 366 367 S p ec ies P eak f re q ue nc y (mea n ± SD, i n kHz) Numb er of pul s es (mea n ± SD) Call dur atio n (s) (mea n ± SD, i n s) N u m be r o f ca ll s i n ca l l se ri es E. [t o be rev e ale d] (N = 15 fr ogs) 5 .2 3 ± 0. 11 7 3. 94 ± 18 .7 8 2 .2 1 ± 0. 54 1 E . na r i ne ns i s (N = 6) 5 .6 4 ± 0. 21 8 .9 8 ± 2. 45 0 .2 8 ± 0. 070 5 –14 E . bo u len ge r i - Gor go n a (N = 6) 4 .7 6 ±0 .11 1 4. 48 ± 2 .19 0 .3 0 ± 0. 036 2 –3 E . bo u len ge r i - Nariñ o (N = 5) 5 .5 1 ± 0. 11 1 3. 09 ± 1 .75 0 .2 8 ± 0. 042 3 –6 E. af f. es pin os ai NA NA NA NA 368 At the typ e locality the ne w species occurs in sympatry with A ndi no bat es min utus b ut differs in b ody 369 size and col orati on (Fi g. 3). A ndi no bat es min utus h as ora ng e rath er th an yell ow sp ots on th e gr oin 370 an d d orsal side of the arms a nd its supral abi al b an d is dark ora n ge rat her th a n w hite as in E. [to b e 371 reveal ed] . V entr ally in A. min utus th e pro p ortion of black a nd tur qu oise blu e is similar. In contrast, in 372 E. [to be r eveal ed] th e col oratio n is mai nly turqu oise bl ue with so me black blotch es (Fig. 2 C). In 373 ad ditio n, ad ults of A. min utus ar e m uch smaller (m ea n = 1 2.0 0 m m, SD = 0.9 1 mm, N = 5) tha n 374 ad ults of E. [to be r eve ale d] (17. 99 m m, S D = 0.9 5, N = 1 6), but A. min utus can be c onf used with E. 375 [to be rev eal ed] juv enil es. Th e color ation of juve nile E. [to b e reve al ed] is very simil ar to E. [to b e 376 reveal ed] ad ults, so color can still be use d to differe ntiate t he tw o sp ecies. 377 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint 378 Fig ure 3. Im ag e s in life of Ep i pe dob a tes [ t o be r e vea l ed ] sp. no v. in c om pari s on wit h clo s e 379 co ng e ner s a nd sy mp atric s pe cie s. A. E . [to be reve ale d] sp. n ov. from the typ e lo cality of 380 La drilleros, V alle d e C auc a, C olom bia ( A ND ES: A:5 26 1; SV L = 2 0.0 mm; a dult m al e; par atype); B. E. 381 narin e nsis from Biot op o, Nari ño, Col om bi a (AN DE S:A: 37 0 4; 16. 39 mm; a dult m ale) ; C. An din ob ates 382 min utus from L a drilleros, Vall e d e C auc a, Col om bia (A ND ES: A:5 26 6; 1 3 m m; sex n ot det ermi ne d); D. 383 E. bo ule n geri from Isla G org o na, C a uca, Col om bia (in dividu al n ot capt ure d); E. E. espin osai fro m Río 384 Pal en qu e, S ant o D omi ng o d e los Tsác hila s, Ecuad or (individ u al not ca ptur ed); F. E. aff. espin osai 385 from L a N utria, Nari ño, C ol om bia (A ND ES :A:24 7 6; 17. 77 mm; sex n ot det ermi ne d); G. E. bo ule n geri 386 from M ara grícola, N ariñ o, Col om bia (A N D ES:A: 24 7 2; 18. 96 mm; a d ult fem ale); H. A side-by-side 387 ima ge of E. bo ul en geri from Isla G org o na (AND E S:A:3 6 95; 2 0.6 5 m m; ad ult fem ale) an d E. [to b e 388 reveal ed] sp. n ov. from Pi an g uita, V alle d e C auca, Col om bia (A ND E S:A:3 69 0; 1 6.4 2 mm; ad ult 389 femal e) d em onstrati ng th e lar ge size differ ence bet we e n the t wo sp ecies. All ima ges were t aken by 390 RD T exce pt for A a nd C, w hich w ere take n by JCR. P h otos are not to scal e. 391 392 M o l ecu l a r an d p hy l oge ne t i c ana l ys es 393 We estim ate d a phylo g eny usin g existing dat a (Ló p ez-Hervas et al. 20 23) an d 1 0 n ew se qu e nces 394 from five E. [to b e reve ale d] from th e type locality. The r esultin g p hylog eny co ntai ns strong su pp ort 395 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint for the m on o phyly of E. [to b e reve ale d] a nd rec overs kno wn phylo g en etic relatio nships a mo ng 396 species in this g en us (Figs. 4 a n d S 2; Ló p ez-Hervas et al. 20 23). T h e sister species to E. [to be 397 reveal ed] is E. n arine nsis , which occurs in the sout h-ce ntral p art of the Pacific coast of Col om bia. 398 To get her, th ese tw o sp ecies constitute a c lad e of E pip ed o bat es that is hig hly diverg ent from t he rest 399 of the ge nus. 400 Th e aver ag e p- distanc es am on g cla des of Epip e do bat es species, inclu din g tw o clad es within t he 401 no min al species, E. b o ule ng eri (from th e type-locality of Isla Gor go n a a nd fro m the mai nla nd) sh ow 402 that E. [to b e reve ale d] is ge netically m ost similar to E. nari ne nsis (1.77 % f or 12 S– 16 S an d 5. 39 % f or 403 CY TB ), as ex pecte d give n th e m olecul ar phylo g eny. 404 405 406 Fig ure 4. Ph ylo ge n etic p ositio n a n d g e netic dist an ce s of E. [to be r ev eal ed] s p. no v. a nd oth er 407 sp eci es of E pip ed ob ate s . A A species-l evel m aximu m-likeliho o d p hylog eny of Epi pe do b ates 408 estimate d in IQ- TR EE usin g two nucle ar g en es ( BM P2, H 3 ) a nd t hre e mitoch o ndrial ge ne fra gm e nts 409 (C YT B , 12 S–1 6S , C R ) (se e Fi g. Su p p. m aterial S 2 for full p hylo ge ny). No de su p por t values from 410 10, 00 0 ultraf ast bo otstrap re plicates in dicate d strong s up port of E. [to be r eveal ed] sp. nov. as the 411 sister-group of E. n arin ensis . B Matrix of me an pairwis e p-dista nces a mo n g specie s of Epip ed o bat es . 412 Th e lo wer tria ngl e distanc es are base d o n CY TB a n d the up p er trian gle dista nces ar e from 12S –1 6S . 413 Epi pe do b ates [to b e reve ale d] is ge netical ly most similar to E. narin e nsis (1.77 % f or 12 S– 1 6S an d 414 5.3 9 % for CYT B ). A bbr eviatio ns: cur, E. [ to be r eveal e d] sp. nov; n ar, E. nari ne nsis ; bou N, E. 415 bo ule ng eri (Nari ño, Col om bia); b ou G, E. b oul en geri (Isla G org on a, Ca uca, Col om bia ); aesp, E. aff. 416 espin osai ; es p, E. espi nosai ; tri, E. tricolor ; mac, E. mach alilla ; a nt, E. a nth onyi . Se e Su pp. m aterial 417 S2 for full p hylo ge ny. 418 419 Th ere are sig nificant differ ences in bo dy size betw e en Epi pe do b ates [to b e reve ale d ] sp. nov., E. 420 narin e nsis, and E. bo ule n geri (G org on a a nd Nariñ o) (Kruskal-W allis test: H 3 = 19.0 58, P < 0.0 01; Fi g. 421 5A). E pip ed o bat es bo ule n geri - Go r go na e xhibits significantly larg er me an bo dy size tha n the oth er 422 po pul atio ns an alyzed. A Wilcoxo n test show ed t hat E. [to be rev eal ed] sp. n ov. is larger th an E. 423 narin e nsis (P = 0.05, N = 33) an d small er tha n E. b oul en g eri -Gor go na ( P < 0.0 06, N = 32). We fo u nd 424 no sig nificant differ enc es in b ody size bet we en E. [to b e reve ale d] sp. nov. a nd t he po pul atio ns of 425 Nariñ o assig ne d to E. bo ule n geri (P = 0. 4 62, N = 33). W e fou n d sexu al dim orp hism in bo dy size 426 (SV L) for E. [to be r eve ale d] sp. nov. (AN OV A: F 1,46 = 6.26 9, P < 0. 01), w her e m ea n SV L in f em ales 427 was larg er (m ea n = 1 8.2 4, SD = 1.0 9 mm, N = 28, T a ble 2) tha n th e me a n in m ales (mea n = 1 7.4 5, 428 SD = 1. 07 mm, N = 20, T a ble 1). 429 E . [t o be reveal ed] sp. nov . sp . n ov . .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint 430 Fig ure 5. Co mp aris on s of S VL an d a d v ertise me nt c all traits bet we en E. [to b e rev eal ed] sp . 431 no v. a nd oth er sp e cie s of Ep i pe dob a tes . A S no ut-vent le ngt h (S VL) B Pe ak freq ue ncy C Call 432 dur ation an d D Nu mb er of p ulses p er call. Ab breviatio ns inclu de: E. bo u - Gor, E. b o ule ng eri - 433 Gorg o na; E. bo u - Nar, E. bo ule ng eri - Na riño; E. n ar, E. n arine nsis; an d E. cur sp. nov., E. [to b e 434 reveal ed] sp. n ov . Black p oints repr ese nt raw dat a, the horizo ntal b ar sho ws the me an, th e sha d ed 435 dia gra m is a smoot he d d e nsity curve showin g the f ull dat a distributi on, a n d the r ectan gle re pres ents 436 the u ncertai nty aro un d th e m ea n usin g a 95 % B ayesia n hig h est de nsity interval. 437 438 Epi pe do b ates [to b e reve ale d] sp. nov., E. narin ensis, an d E. bo ule ng eri (from G org on a a nd Nari ño) 439 display n ote wort hy distinctions in call p ara meters. W e fou n d significant differe nces in the pe ak 440 frequ e ncy of advertise me nt calls am on g a ll species an d p op ulati ons teste d (Kruskal -Wallis test: H 3 = 441 10 9.2 5, P < 0. 00 1; Fi g. 5B). Wilcoxo n test show ed t hat Ep i ped ob a te s [to b e reve ale d] sp. nov. calls 442 with a lo w er pe ak freq u ency tha n E. n arin ensis (P < 0.0 01, N = 81) an d E . bou l en ger i -Nariño ( P < 443 0.0 01, N = 76), b ut its peak fre qu ency is high er tha n for E. b o ule ng eri -Gor go n a (P < 0.0 01, N = 79). 444 We fo un d statistically significant differe nces in call dur atio n (Kruskal-W allis test: H 3 = 95.3 0 2, P < 445 0.0 01; Fi g. 5 C). Epi pe do b ates [to b e reve ale d] sp. nov. exhi bits lon ger calls tha n E. narin e nsis (P < 446 0.0 01, N = 81), E. b o ule ng eri -Nari ño ( P < 0.0 01, N = 76) a n d E. b oul en g eri -Gor go na (P < 0.00 1, N = 447 79). Simil ar results wer e fo un d for th e n u mb er of p ulses per call (Kr uskal-Wallis test: H 3 = 107. 52, P < 448 0.0 01, Fi g. 5 D). Epi pe do b ates [to b e reve ale d] sp. nov. h as calls with mor e p ulses tha n E. n arin ensis 449 (P < 0.0 01, N = 81), E. b o ule ng eri -N ariño (P < 0.0 01, N = 76), a nd E. b oul en geri -G o rgo na (P < 0.0 01, 450 N = 79). 451 452 De scri ption 453 454 Col oratio n of h olot yp e in life (Fig. 2A–D ) . Dorsal surfaces ar e d ark brow n wit h an obliq u e later al 455 stripe exte n din g fro m th e p osterior re gi on of t he ey e to th e groin, with m etal lic oran g e-yellow 456 coloratio n in the ant erior regi on b eco mi ng m ore yello w tow ards the groin. Gr oin regi on is dark 457 bro wnish black with a distinct yellow blotch conti nui ng to t he ant erior conc eal ed sur face of th e thig h. 458 Fla nks are bl ack. Forelim bs a nd hin d lim bs are dark bro w n. Ant erior re gio n of th e up per ar m h as a 459 [ to be r ev ea l ed] [to b e r ev ea le d] .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint yellow bl otch similar i n col or to th at of th e thi gh. S upr ala bial strip e is cre amy whit e, exte n din g fro m 460 the n ares to th e axilla. V e ntral surfaces ar e turq uois e blu e wit h irreg ular bl ack spots. Iris i s copper. 461 462 Col oratio n of h olot yp e i n pr es erv ativ e (Fig. 2E –K; 7 0% eth a nol). D orsally bl a ck to dark bro wn, 463 hin d lim bs dark bro wn. Lig hter br ow n f orel imbs with so m e dark bro wn bl otches. Yell ow bl otches i n life 464 on for elimbs as w ell as inn er a nd dorsal t hig hs are w hite in preservati on. V entr ally pal e w hite to d ark 465 gray with r ou n de d bl ack to d ark bro wn s p ots. Groin black. Obli qu e lat eral lin e is d a rk gray exten din g 466 from th e p osterior re gio n of t he eye to t h e gr oin an d ca n b e very faint. Li ght gray supral abi al b an d 467 run nin g from th e tip of th e face t o the axilla. Tw o el on g ate d black spots in th e g ular regio n (Fi g. 2 F). 468 469 Col oratio n of ty pe s eries a nd ot her po pul ation s i n life. All i ndivid u als in th e ty pe s eries ex hibit a 470 u n i f o rm d a r k b r own c o lo r o n t he d o r s um a nd d o r s a l s u r f a ce s o f l i m b s ( S upp l . ma t e r i a l S3 ) . The 471 dorsu m, he a d, thigh, an d sha nks featur e a skin texture covere d with fin e, eve nly distributed tu b ercles. 472 Fla nks an d c onc eal ed s urfaces of th e f orelim bs a n d hi n dlimbs are smo oth a n d solid bl ack. All 473 individ uals sho w a n obli qu e later al stripe that stretches from the p osterior re gio n of the ey e to the 474 groin. It a pp ears met allic ora ng e-yello w in th e ant erior re gio n a nd gra d ually tra n sitions to a m ore 475 yellow col or as it reac hes th e groin. Th e obliq u e lat eral stripe differs slightly am o ng po p ulatio ns. In 476 the L adriller os p op ulati on (type locality), the o bliq ue lat eral stripe dis plays a n or ange - y e l l ow c o lo r w i th 477 indistinct ed g es an d stretches fro m the f ront p art of th e gr oin to t he eye, gr ad ua lly changi ng fro m 478 yellow t o a mor e br ow nish-or an g e h ue. Th e stripe co ntin ues ont o th e u p per eyel id a nd f ad es as it 479 reach es th e ca nth us rostralis. In t he A n chicayá po pul atio n, th e obli qu e lat eral s tripe tak es o n a 480 yellowish h ue a n d, in certai n sp ecim ens, it may ta per, fra gm e nt int o patch es, an d gr ad ually disa pp ear 481 bef ore r eac hin g th e eye. I n so me in dividu als from Pia n guit a, th e obli qu e l ateral stripe exhi bits re gul ar 482 interru ptio ns alo ng its entire l en gth. 483 484 In all individ u als of E. [to be r eve ale d] sp. nov., the groi n regi on is char acterized by a d ark brow n-485 black color, with a n oticea ble yell ow bl otch that in s om e individ u als exten ds to the fr ont in ner thi g h 486 (Su ppl. m aterial S3). M ost individ uals h ave a p aracl oacal sp ot similar in color atio n, but sm aller a nd 487 more elo n gat ed, o n th e p osterior d orsal surface of th e thi gh. In t he A nchicay á p op ul ation, th ese sp ots 488 are pr ese nt in o nly some i ndivid uals. M ost individu als h ave a yell ow patch on th e d o rsal regio n of th e 489 arm th at match es the c olor of th e conc eal ed thi gh patc h. All individ uals ex hibit a cre amy w hite to pal e 490 turqu oise blu e u pp er labi al stripe th at is nota bly lighter th an t he obli qu e later al stripe. In som e cases, 491 this stripe has a slig ht iridescent qu ality and exten ds from b elo w th e n ostril to the a xilla. Also, in 492 certain in dividu als, it continu es p osteriorly as a vag uely d efin ed ve ntrol ateral stripe. Th e ventr al 493 surfaces of the t hro at, belly, an d thi ghs ex hibit a pal e turq uois e blu e to white col or w ith irregul ar black 494 spots an d p atter ns resem blin g w orm-like lines (vermicul ations). In m ales, this colorat ion m ay b e 495 partially o bscure d or interr upt ed by a diffu se dark gr ay pig me nt locate d just a nterior to the pector al 496 regio n a n d the v ocal sac. A few i ndivid ual s display a diffuse yell owish c olorati on to war ds the o uter 497 ed ges of th e b elly. 498 499 Vo calizatio n s. E pip ed ob ates sp ecies h ave p ulse d calls. Advertise me nt calls are co mp ose d of 500 gro ups of calls, on e call for E. [to be r eveal ed] sp. n ov., 2– 3 for E. b oul en g eri -Go rgo na, 3– 6 for E. 501 bo ule ng eri -N ariñ o a n d 5– 14 for E . na r i nens i s (Fig. 6). Calls of E. [to b e r eveal ed] sp. n ov consiste d of 502 2 2– 122 p u l s e s pe r c a l l (m ea n = 73 . 98 , SD = 18 . 77 , N = 15 ) , w i th a c a l l du r a t i on o f 0 . 67 –3 . 88 s (m ea n 503 = 2.2 1, SD = 0.5 4 s, N = 1 5). Th e p eak fr eq ue ncy ran ge d b etw e en 4.9 8 a nd 5.4 6 kHz (me an = 5.2 3, 504 SD = 0.1 1 kHz), an d th e freq ue ncy inter q uartile ba nd widt h b etw e en 0.2 1 a nd 0.6 7 kHz (mea n = 0. 35, 505 SD = 0.8 7 kHz) (Tabl e 4). We recor d ed two m ales close to a fe mal e with vocalizat ions differe nt from 506 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint their a dvertisem ent call (Fig. 6E). T hes e vocalizatio ns, most likely a courtship call, ran ge d from 0.2 1–507 0.3 1 s in len gth (m ea n = 0. 25, S D = 0.0 3 s, N = 2), and incl ud ed 7– 9 p ulses p er call (mea n 8. 25 ± SD 508 0.8 3). Th e p eak fre qu ency ra ng ed bet we e n 4.7 8 –5. 17 kHz (m ea n = 4.9 6, S D = 0.1 3 kHz). 509 Ta ble 4. S um mary statistics of spectral an d te mp oral ch aracteristics of the adve rtisement calls of 510 Epi pe do b ates [to b e reve ale d] sp. nov. 511 512 Par am et er s Call Dura tio n (s) Num be r of p u l se s Puls e rat e Int erc all int erval (s) Puls e dur ati o n (s) Int er puls e i nt erval (s) Low F r e que nc y (kHz) High F r e que nc y (kHz) Pe ak F r e que nc y (kHz) B and w i dt h 90 % (kHz) M e an 2 .2 13 7 3. 938 34 .8 9 4 6 2. 675 0 .0 17 0 .0 13 4 .8 45 5 .5 86 5 .2 37 0 .3 54 S D 0 .5 41 1 8. 786 2 .0 77 6 0. 762 0 .0 04 0 .0 09 0 .2 22 0 .2 10 0 .1 15 0 .0 87 M i n 0 .6 74 2 2. 000 26 .0 0 0 1 5. 120 0 .0 09 0 .0 01 4 .2 36 5 .2 21 4 .9 81 0 .2 15 Max 3. 88 8 12 2. 0 0 0 39 .0 0 0 31 5. 0 1 2 0 .0 28 0 .0 68 5 .2 87 6 .0 93 5 .4 69 0 .6 75 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint 513 Fig ure 6 . Vo calizatio n s in E. [to b e re ve ale d] sp. no v. a nd oth er sp e cie s of Ep ip ed oba t es . 514 Sp ectrogr ams (a bove) an d oscillogr ams (bel ow) sh owi ng ge n eral asp ects of the structure of on e 515 spont an e ous a dvertisem e nt call for on e m ale of A E. [to b e reve ale d] sp. n ov. of La d rilleros, Valle del 516 Ca uca, B E. nari ne nsis of Bioto p o, Nari ño , C E. bo ule n geri -G org on a (Typ e loc ality) an d D E. 517 bo ule ng eri -N ariñ o of M ara grícola, N ariñ o. Advertise me nt calls consist of call grou pin gs, comprisin g a 518 [ t o be r e v eal ed ] [to be revealed ] sp. nov . [t o be re v e aled] sp. nov . .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint single call for E pip e do bat es [to be r eve ale d] sp. nov., 2 –3 calls for E. b oul en g eri -Go rgo na, 3 – 6 calls 519 for E. bo ule n geri -N ariñ o, a nd 5– 14 calls for E. n arine nsis . E Sp ectrogr ams a n d oscillogra ms sho win g 520 ge ner al asp ects of the structure of on e co urtship call of E. [to b e rev eal ed] sp. n ov. of La drilleros, 521 Valle del Ca uca. F Co mp ariso n of o ne ad vertisment call b etw ee n E. [to b e reve ale d ] sp. nov. an d 522 oth er species of Ep i ped ob a te s . E. bo u - G or: E. bo ule n geri - Gor go n a, E. b ou - N ar: E. bo ule n geri - 523 Nariñ o, E. n ar: E. n arin ensis an d E. cur sp . nov.: E. [to be rev eal ed] sp. n ov . Fast F o urier Tra nsform-524 FF T = 2 56, ov erla p = 9 0 %. 525 526 A princip al com p on ent an alysis (PCA) indi cated th at th e first three princip al com p on ents expl ain 88 % 527 of the vari ation i n calls (Fig. 7 A). PC 1 expl ain ed 45 % of th e variatio n a nd was p ositively associate d 528 with call d uratio n, n um ber of pulses, a n d intercall interv al. PC 1 w as also n eg atively relate d to hi g h 529 frequ e ncy an d 9 0 % b a nd widt h (Su p pl. ma terial S4). P C 2 expl ain ed an ad ditio nal 33 % of the vari atio n 530 in call par am eters. It was positively relate d to lo w freq ue ncy an d p e ak frequ e ncy. PC3 explai ne d 1 0 % 531 of the vari ation an d w as p ositively associated wit h freq ue ncy interq u artile b an dwi dth 90 % ( Fig. 7 A, 532 Su ppl. m ateri al S4). In su mm ary, PC 1 wa s mainly associate d wit h tem por al char acteristics of the 533 advertise me nt call an d P C 2 with sp ectral characteristics. The lin ear discrimina nt an alysis (LDA) (Fig. 534 7B) fu nction using five P Cs sho w ed th at E . [to be reve ale d] can be easily differe ntiat ed fro m oth er 535 species b ase d o n their a dvertisem e nt calls. LD1 w as p ositively associated wit h P C1 (temp oral 536 characteristics) (LD coef. = 1.7 1), an d L D 2 w as ne gatively associat ed with P C2 (sp ectral 537 characteristics) (LD coef. = -1.4 7). This m ea ns E. [to b e reve ale d] is distinguish e d b y having l on ger 538 calls and a gre ater nu mb er of p ulses (Figs 7B a n d 5 C-D). With resp ect to low a nd p eak freq u ency, E. 539 [to be rev eal ed] calls are ch aract erized by interm edi ate val ues com p are d to th e oth e r species 540 an alyzed (p e ak frequ e ncy: 4.84 ± 0.2 2 kH z, N=15). E pip ed o bat es bo ule ng eri- G org o na has th e 541 low est pe ak freq ue ncy (4.76 ± 0.1 1 kHz, N=5) a n d E. n arin ensis has th e hi gh est pe ak frequ e ncy 542 (5.64 ± 0.2 1 kHz, N= 6) (Figs 7 B a nd 5B). 543 544 545 Fig ure 7. Differen c es i n the ac o ustic pa ramet ers of E. [to b e rev eal ed] sp. n o v. co mp are d to 546 oth er sp e cie s of E pip ed o bat es . A Th e relative co ntributi on of eac h aco ustic variable to th e first five 547 princip al com po ne nts obt ain e d in th e Prin cipal C om po ne nt A nalysis. The abs olut e valu e of e ach 548 contrib ution is re prese nte d accor din g to th e size of the circle, whil e blu e a n d red col ors show positive 549 an d n eg ative co ntributi ons, resp ectively. Call d urati on, n um b er of p ulses an d int ercall interval ar e 550 [t o b e r ev eal e d] sp . n ov. .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint strongly a nd positively associate d with PC 1 w hile hi gh fre q ue ncy an d 9 0 % b a nd widt h are stro ngly 551 an d n eg atively associate d wit h PC 1. P C2 is strongly an d p ositively related to l ow fre qu ency a nd pe ak 552 frequ e ncy, while th e freq u ency inter qu artile b an dwi dth 90 % is n e gatively associate d with P C2. B A 553 Lin ear Discrimin ant An alysis using PCs a s input in dicates E. [to be rev eal ed] ca n b e differe ntiat ed 554 from oth er sp ecies bas ed on th eir a dvertisem ent calls, particul arly regar din g call dur ation an d n u mb er 555 of puls es (PC1 l oa ds p ositively on L D1). Dots indic ate th e in dividu als, color indicat es the sp ecies, 556 an d ellips es indicat e the 9 5 % co nfide nce i ntervals for L DA dat a p oints. 557 558 E t y mo l ogy 559 Th e specific epit het of this n ew s pecies re fers to the m usical ge nre t hat ori gin ate d o n the so uth ern 560 Pacific coast of Col om bia an d Ecu a dor, w her e E. [to b e reve ale d] occurs an d als o contrib utes to th e 561 local sou ndsca p e. [to be r eveal e d] , also kno wn as ba mb uco viej o , is an Afro- Col om bia n sou n de d 562 practice that ins pires d ancin g a n d transmi ts the ha ppi n ess and c ultural tra dition of this regio n a n d is a 563 symbol of resilie nce in th e face of racial a nd re gio n al o ppr ession (A ba día, 1 9 73; Ari stizabal, 20 0 2; 564 Biren b au m Q uinter o, 2 00 6, 2 01 9). We na me d this species in ho nor of, an d as a n h om ag e to, this 565 musical g enr e th at repr esents th e culture of the so uth ern Col om bia n P acific becaus e: “la música, 566 como l a vida, n o se p u ed en dej ar p erd er” , which tra nslates to “m usic, like life, canno t be all ow ed to 567 be lost” (Cruz H oyos, 2 01 6). It is a nou n in a pp osition of masculi ne ge nd er. 568 569 Distrib ution 570 Epi pe do b ates [to b e reve ale d] sp. nov. oc curs in the D ep artm ent of Valle d el Ca uca in the P acific 571 lowla n ds of sout hw estern Col om bia. T h ese frogs in ha bit lowl an d for ests from 0– 26 0 m. Th e type 572 locality is Ladrilleros, B ue nav ent ura, V alle del C auc a, Col om bia. W e als o o bserved t he sp ecies in 573 are as close to the typ e locality inclu din g C orregi mie nto Pia ng uita a n d C orregi mie nto Juan Ch aco (L a 574 Barra be ach) in th e m u nicipality of Bu e na ventur a. Th e distrib utio n tow ards th e w estern fla nk of the 575 wester n mo u ntai n ran g e is in the V ere d a El Da n ubi o, the up per basin of the Anc hicayá River, D a gu a, 576 Valle del Ca uca. If we assum e th at the col oratio n traits of the ne w speci es are co nsistent for all 577 po pul atio ns of this species, records fro m the iN atur alist Platform wo uld exten d th e d istribution of E. 578 [to be rev eal ed] 19 4 km (13 2 km) straig ht line so uth to th e m unicip ality of Tim biq uí, Ca uca (see 579 iNatur alist observati ons N o. 1 35 25 3 84 3 a nd 85 21 4 43 9 a nd Fig. 1). B eca use in divid uals of this 580 species w ere pr evio usly assigne d to E. b o ule ng eri , we rec om me nd f urth er explor ati on an d insp ection 581 of mus eu m specim ens to bett er un d erstan d the ge o gra phic distributi on of this specie s. 582 583 Ec olo gy 584 Epi pe do b ates [to b e reve ale d] sp. nov. is a terrestrial species fo un d o n th e gr ou nd durin g th e d ay in 585 agr oforestry are as, on th e e d ges of seco n dary forests, or in small p atch es of disturb ed sec on dary 586 forest alw ays ne ar or withi n m arshes a n d/or slow-flo win g streams (Fi g. 8). Th e sp ecies is also likely 587 prese nt in prim ary forest in th e regi o n. We observe d in dividu als actively movin g am o ng th e gr ass an d 588 leaf litter, or actively calling at th e e dg es o f water b o dies. Th e typ e locality (La drilleros, Valle de 589 Ca uca, C olo mbi a) inclu des tiny forest frag me nts am on g h um an dw ellin gs. Usually, these are as are 590 conta min ate d with gar ba g e or a gricultural residu es (Fig. 8 A– F). In A nchicayá, po pul ations of E. [to be 591 reveal ed] are us ually fou n d alo n g roa dsid es an d forest e d ges, w he nev er small streams a nd l eaf litter 592 are pr ese nt (Fig. 8 G–J). W e o bserved gre ater vocal activity durin g the mor nin g a nd in the l ate 593 aftern oo n th an duri ng war mer or su nni er p arts of the d ay. 594 595 Th e d ensity of E pip ed ob ates varies acr oss species an d sites in Col om bia . D urin g o ur fieldw ork, we 596 notice d th at the po pul atio ns of E. b oul en g eri at Isla Gorg o na an d M ara grícola ar e q uite a bu n da nt. For 597 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint exam ple, m ore t ha n five individ u als can b e h ear d calling at the s am e time, so it was very difficult to 598 record a sin gle mal e. F or E. n arine nsis at Bioto p o n atural res erve in dividu als wer e calling l ess 599 de nsely b ut wer e still abu nd a nt, with 8- 10 m distanc e b etw ee n callin g m ales. But in oth er ne arby 600 locatio ns we have he ard only tw o or thr ee individu als in a 3– 4-h our walk. T he in dividu als of E. [to b e 601 reveal ed] sp. n ov. are mor e spre ad out a n d mal es w ere o bserve d to call at a p proxi mately 4 t o 5 m 602 distance fro m e ach oth er at type loc ality ( La drilleros a nd La Barr a). At Anc hicaya, mal es call every 3-603 4 m a nd are associate d with sm all stream s at the e dg e of th e u np ave d ro ad. W e d o not kno w th e 604 reaso n for differ enc es in d ensity, as the p attern do es not nec essarily correspon d to differe nces in 605 ha bitat q uality. Whil e Isla Gor go na is a na tional park, E. b oul en g eri ap pe ars to b e m ore a b un da nt in 606 the distur be d ar eas a djac ent to fiel d statio n b uildin gs. Th e M ara grícola site w h ere w e sam ple d E. 607 bo ule ng eri in N ariñ o is an experi me ntal ca cao pl ant atio n. La drilleros a n d L a B arra ar e disturb e d sites 608 similar to wh ere E. bo ule n geri w as a bu nd ant at Isla G org on a, yet E. [to b e reve ale d ] did n ot occur at 609 such hig h d e nsities. It is plausibl e that th e E. [to be rev eal ed] is mor e vuln era ble to ha bitat 610 transform atio n tha n E. bo ule ng eri , but ad d itional res earch is n ecessary to u nd erstan d w hat drives 611 differe nces in d ensity across sites and sp ecies. 612 613 Unf ortun ately, w e di d n ot collect larval stages of E. [to be rev eal ed] . L arval m orp hol ogy of 614 De ndr ob atoi de a h as contrib ute d to un ders tan din g of th e p hylog e netic relatio nshi ps within th e 615 superf amily (An ga n oy-Criollo & C ep ed a-Q uilind o, 2 01 7). It wo uld b e int erestin g to collect tad pol es of 616 the n e w species for co mp arison with ot her Epip e do bat es species. C om pariso ns of la rval stages 617 ma de by An ga n oy-Criollo an d C ep e da- Qu ilindo ( 20 17) det ected so me differe nces b etw ee n th e 618 po pul atio ns of Ecu ad or a nd so uth w est of Col om bia in bo dy sha p e, tips of mar gin al pa pilla e, an d 619 patter n of tail color atio n. Ho w ever, the sp ecime ns from C olo mbi a us ed in t heir stud y likely correspo nd 620 to E. b oul en geri -N ariñ o ( E. b oul e ng eri sen su stricto ) and w e are not certai n of th e id entificatio n of th e 621 specime ns from Ecua d or, which co uld cor respo nd t o E. aff. espi nos ai , E. bo ule n geri sensu stricto , or 622 E. espin osai , w hich all occur in the r egi on (Ló pez-H ervas et al., 2 02 3). 623 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint 624 Fig ure 8: H abit at struct ure of Epi pe d ob ate s [to b e rev eal ed] s p. n ov. at tw o loc alities. A– C 625 Imag es of th e typ e loc ality at L adriller os , Bu en ave ntur a, V alle d el C a uca, C olo m bia. Usu ally, this 626 species is foun d o n ro adsid es ne ar strea ms forme d by rainf all. D– F At th e type loc ality, we obs erved 627 frogs in h abitats co ntami nat ed with g arb a ge or agricultur al w aste. Not e th e frog in F (red circle). G–J 628 Ha bitat c har acteristics in Anc hicayá, Da g ua, V alle d el C a uca, Col om bia. Ima g es B, C a n d E wer e 629 taken by RD T, all ot hers by M BC. 630 631 Co ns erv atio n st atu s 632 Th e p op ulati ons o bserve d in th e fo ur local ities (Ladriller os, Pian g uita, L a B arra, a nd Anchic ayá) are 633 pro ba bly affecte d by h ousin g co nstruction , garb ag e, or a gricultur al waste as w ell as forest 634 fragm ent atio n a nd l oss of ha bitat. We do not kn ow if thes e p op ulati ons h ave ad apt e d to h um an 635 disturb ance or if they are r em na nts of the origin al distributi on of the sp ecies, but co n ge ners in 636 Ecua d or are ab un d ant in hig hly mo dified ha bitats such as caca o a nd ba n an a p op ul ations (th ou g h 637 they are not ably a bse nt from Africa n p alm plant atio ns; RD T p ers. obs.). Altho ug h w e consid er th at E. 638 [to be rev eal ed] sp. n ov. is abu n da nt at th e type loc ality, as three of us (R DT, M B C, JCR) capt ure d 1 7 639 individ uals in a n a p proxim ate ar ea of 3 0 m 2 in four h o urs, we d o n ot kno w th eir ab u nd anc e in less 640 disturb ed ha bitats. Improvin g o ur u nd erstan din g of its conservatio n status will re qui re mo nitorin g a n d 641 explor atio ns in p ote ntial localities in C a uca a nd Valle del Ca uca, es pecially in pr ote cted are as close 642 to the loc alities rep orte d in this study. 643 644 Despit e the l ack of certainty reg ardi ng t he distribution of E. [to b e reve ale d] sp. n ov., its extent of 645 occurrenc e is likely below 20, 00 0 km 2 . We know th e sp ecies occurs in at le ast four localities 646 (La drilleros, Anc hicayá, Pi an guit a, a nd La Barra), with on e m ore pe ndi n g g en etic validatio n 647 (Timbi quí). T he f orests in the ra n ge of this species hav e b ee n, a nd pro ba bly will con tinue t o b e, 648 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint subject to stron g d efor estatio n pressur es that re duc e the qu a ntity and qu ality of avai labl e h abitat an d 649 increase its frag me ntatio n. T hus, w e w oul d reco mm en d cate gorizin g E. [to b e reve a led] sp. n ov. as 650 Vul ner abl e (VU: B1 a, biii; IUC N, 20 1 9). T he distributio n of E. [to b e reve al ed] sp. n ov. is very close to 651 the P arq u e N acion al N atural Ura mb a B ah ía Mál ag a, b ut we hav e o nly record ed t he species in ar eas 652 close to h um an settle me nts. We are n ot a war e of th e pres enc e of E. [to b e reve ale d ] sp. nov. within 653 the p ark. Ho wev er, this conservatio n ar ea protects mor e tha n 4 7,0 0 0 h a of m arin e a nd co astal ar eas, 654 so it is very likely that E. currala o sp. n ov. is foun d withi n the park. M ore rese arch a b out this speci es’ 655 distributio n, ecol ogical re q uirem e nts, and po pul atio n dyn amics will h elp co nfid ently assign it to a 656 threat cat eg ory. 657 658 DISC U SSIO N 659 660 We d escribe Epi pe do b ates [to b e reve ale d] sp. nov. as a ne w speci es fou nd alo ng t he Pacific 661 choco an r ainfor est of Col om bia, which wa s previously consid ere d to b e p art of th e E . boul en g eri 662 species com plex (L óp ez-H ervas et al. 2 02 3). We d escribe this sp ecies as ne w b ase d o n its distinct 663 advertise me nt call, coloratio n, a nd diverg ence fro m its sister species, E. narin ensis . More i nform atio n 664 will be necessary to ev alu ate th e co nservation stat us of E. [to b e reve ale d] , but we pro pose t hat it 665 may b e consi der ed as Vul ner abl e u nd er IUC N cate gorizati on given its relatively small ran ge. 666 Altho u gh E. [to be r eveal e d] is not kno wn to be sym patric with ot her sp ecies of E pip ed ob ates , re gio ns 667 in sout hw estern Col om bia (es pecially e ast of Gu api) h ave not b e en extensively surveyed an d m ay 668 contai n sites with symp atry betw ee n E. [to be reve al ed] , E . bo u len ge r i , an d/or E. aff. espin osai . 669 Epi pe do b ates [to b e reve ale d] d oes occur in sympatry an d ca n b e conf use d with A n din ob ates 670 min utus u nless color p atterns ar e caref ully distinguish ed (se e Di ag nosis section ab ove). Th e ot her 671 species of th e E pip ed ob ates ge nus ar e fo un d al on g th e w estern co ast an d A nd e an foothills in 672 Ecua d or an d P eru. Altho u gh on e speci es ( Epip e do bat es mac ulatus ) is tho ug ht to oc cur in wester n 673 Pa na ma (Gr ant et al., 20 1 7; Jun gfer, 2 01 7), its assignme nt to this g en us has b e en bro ug ht into 674 qu estion ( Ló pez-H ervas et al. bi oRxiv). 675 676 We fin d that th e a dvertise me nt call of E. [to b e reve ale d] is uniq u e com par ed to oth e r Epip ed o bat es 677 distribute d in C olo m bia. Pr eviously, Lötter s et al., (200 3) rep orte d a dvertisem ent cal l differenc es 678 bet we en E p ip edo ba t e s bo u len ge r i record ed at Anc hicayá (n ow E. [to b e reve ale d] ) an d at Lit a (no w 679 E. aff. espin osai ), sug gestin g at th e tim e that th e p op ulati ons m ay b elo ng to a speci es compl ex. In 680 our recor din gs, we f ou nd t hat E. [to be rev eal ed] m akes o nly on e call p er b out, alth o ug h L ötters et al. 681 (20 03) d escribe calls with up to t hre e n ote s; how ever, they recor de d o nly o ne m ale. In the 682 oscillogra m a nd sp ectrogr am of the advert iseme nt call rep orte d in th eir study (Fig 4, Lötters et al 683 20 03), th e inter nal i ntervals are so s hort that it is difficult to define wh e n o ne not e e nds a nd t he next 684 be gins, th us we co nsid er that th ese calls correspo n d to o ne call. 685 686 We pr ovid e d ata o n th e courtshi p call of E . [to be reve ale d] , whic h to o ur kno wle d ge is the first 687 courtship call re port ed for Epi pe do b ates . Like courtshi p calls of oth er n eotro pical p o ison frogs, it is 688 low er freq ue ncy tha n the adv ertiseme nt call of E. [to b e reve ale d] (Cal deir a C osta e t al., 200 6; 689 Go nzález-S a ntoro et al., 20 2 1; Mar qu es Correi a d a R och a et al., 2 01 8; M oss et al., 202 3). It was also 690 shorter a nd ha d fe wer pulses (7. 9 vs 22-1 22 per call). 691 692 Epi pe do b ates [to b e reve ale d] is nam e d in ho nor of th e Afr o-C olom bi an [to be rev eal ed] m usical 693 ge nre an d traditi ons th at arose fro m g e ner ations of multicultur al practices in th e P acific region, 694 starting with African slaves br ou ght to wor k in the g old mi n es, an d conti nui ng to d ay with infusi on of 695 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint ne w id eas a nd i nter pretati ons (Bire n ba um Quint ero, 2 0 19; G uev ara C ald eró n & Go doy Acost a, 696 20 15). As p art of th e cultural herita ge of B lack commu nities in th e P acific region, [to be rev eal ed] 697 music, also kno wn as ba m buco viej o , h elp ed bolster ar gu me nts that le d to th e reco g nition of the l an d 698 rights an d cultur al ide ntity of Afro-C olo mbi ans thro u gh La w 7 0 of 1 9 93 (Bir en ba u m Qui ntero, 20 19). 699 700 A CK NO WL ED GE ME NTS 701 702 RD T a nd MB C tha nk Cristian Flór ez Pai, Luis Alfre do Esteves M en eses, Fray Arria ga, S a ndr a V. 703 Flech as, Da niel Nastac uaz a nd Nat aly Po rtillo for assistance in field work. Ad olfo A mézq uita 704 facilitated th e p ermittin g proc ess in 20 1 4 an d 2 01 6 a n d Mireya Osorio R. in 2 02 2. We als o tha nk 705 AN DE S, QC A Z, an d M VZ muse u m p erso nn el inclu din g A ng ela Sá nch ez G. an d C a rol Sp enc er for 706 their assistance in accessioni ng sp ecime n s. We tha nk Val eria R amír ez-Casta ñ ed a for assistance 707 with P CR. W e th ank M auricio Rivera for a ccess to the specim ens of th e Instituto d e Cie ncias 708 Nat urales at Universid ad Naci on al d e C olom bia, B og otá, an d N ataly C asas for h er hel p in takin g 709 ph oto gra phs of muse um s pecim ens. 710 711 Ad dition al infor matio n 712 713 Co nflict of inter est 714 Th e a uth ors hav e d eclare d that no co mp e ting int erests exist. 715 716 Ethic al st atem e nt 717 No ethical state me nt w as rep orte d. 718 719 Fu ndi ng 720 Fin ancial su p port for this proj ect to RD T w as provid ed by start-up fu ndi ng fro m U niversity of 721 Califor nia B erkel ey an d gr ants from th e S ociety of System atic Biolo gists, North C arolin a 722 Her pet olo gical S ociety, Society for the Stu dy of Re ptiles a nd Am phi bia ns, Chica g o Her pet olo gical 723 Society, T exas Her pet olo gical S ociety, the EE B Pr ogr am at Univ ersity of Texas at Austin, N atio nal 724 Scienc e F ou nd atio n Gra d uat e R esearc h Fell owshi p Pr ogr am Gr ad u ate R ese arch Op portu nities 725 World wid e, N atio nal G eo gra p hic Yo un g E xplorer Gr ant. S up p ort was also provid ed by NS F to D CC 726 a nd RD T ( DEB 15 56 967 ) . 727 728 Aut hor c o ntributio n s 729 Fu ndi ng acq uisition: R DT, DC C. C onc e ptu alization: MB C, R DT. Investig atio n: MB C, RD T, JCR. 730 Meth o dol ogy: M BC, R D T, JCR, AJ C. Fo rmal a nalysis: MBC. Writin g – origi nal d raft: MBC, R D T. 731 Writing – r eview an d e ditin g: MB C, RD T, DC C, JCR, AJC. 732 733 Aut hor O RCI Ds 734 Mileidy B eta nco urth-C un d ar https://orcid.o rg/00 0 0-0 00 3-2 36 8-6 0 28 735 Juan Ca milo Ríos-Orju ela https://orcid.org /00 00- 00 01- 69 7 6-9 13 1 736 An dre w J. Crawf ord htt ps://orcid.org/ 00 00 -00 03- 31 53- 6 898 737 David C. Ca n nat ella htt ps://orcid.org/0 0 00 -00 01- 86 75- 05 2 0 738 Re becca D. Tarvin https://orcid.org/ 00 0 0-00 01- 53 8 7-7 25 0 739 740 Dat a a vaila bility 741 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint All of the d ata th at supp ort the fin din gs of this study are availa ble in th e mai n text a nd w ere upl oa de d 742 to pu blic dat ab ases. 743 744 R EF ER EN CES 745 746 Ab adí a G (1 97 3) L a m úsica folklórica colo mbi an a. Univ ersida d N acion al d e C olo mbi a, Dirección de 747 Divulg ación Cult ural, B og ota D.C.- C olom bia, 1 58 pp. 748 An ga n oy-Criollo M, Ce pe d a-Quili nd o B (2 01 7) R ed escription of the ta d pol es of E pip ed ob ates 749 narin e nsis and E. b oul en geri (A nur a: D en dro batid a e). 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It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint Sa ntos JC, B aq uer o M, B arrio-A mor ós C, Col om a L A, Erdtm a nn LK, Li ma AP, C a nn atella DC (2 0 14). 879 Ap osem atism incre ases aco ustic diversifi cation an d speci ation i n p oison fro gs. Pro ceedi n gs of 880 the R oyal S ociety B, 2 81, 20 14 1 76 1. https ://doi.org/ 10. 10 98/rsp b.2 0 14. 17 61 881 Sa ntos JC, C an nat ella D C (2 01 1) P he n otypic integrati on em erg es from a p osem atism an d scale i n 882 poiso n fro gs. Proce edi ngs of th e N atio nal Acad emy of Scienc es of the Unit ed States of 883 Am erica, 10 8(1 5), 6 17 5– 6 18 0. https://doi. org/1 0.1 0 73/ pn as.10 1 09 52 1 08 884 Sa ntos JC, C olo ma LA, C an n atella DC (2 00 3) Multi ple, recurrin g ori gins of a p osem atism an d di et 885 specializatio n in p oiso n frogs. Pr oce edi ng s of the N ation al Ac ad emy of Sci ences of the U nite d 886 States of Am erica, 10 0(2 2), 1 27 92 – 12 79 7 . https://doi.org/ 10. 10 7 3/p nas.2 13 3 52 11 0 0 887 Sa ntos JC, C olo ma LA, S um mers K, C ald well JP, R ee R, Ca n nat ella D C (2 00 9) Am azoni a n 888 am phi bia n diversity is primarily derived fro m late Mi oce ne An de a n line a ges. P Lo S Bi olo gy, 7 889 e1 00 0 05 6(3), 0 44 8 –0 46 1. https://d oi.org/ 1 0.1 37 1/jo urn al.p bio.1 0 00 05 6 890 Su eur J, Au bin T, Sim o nis C (20 08) Se ew ave, a fre e m od ular to ol for sou n d a nalysis and synth esis. 891 Bio acoustics, 18, 2 13 – 22 6. http://cran.r-pr oject.org/src/contrib/D escriptions/ 892 Tarvin R D, P ow ell E A, Sa ntos JC, R on S R, Ca n nat ella D C (2 01 7) T he birth of a po sematism: Hi gh 893 ph en otypic diver ge nce a n d low ge n etic diversity in a youn g clad e of p oiso n frogs. M olecul ar 894 Phylo ge n etics and Evoluti on, 1 0 9, 28 3 –2 9 5. https://doi. org/1 0.1 0 16/j.ymp ev.2 01 6.1 2 .03 5 895 Ve na bles W, Ri pley B ( 20 02) M o der n A pp lied St atistics with S (Fourt h E dition). S pring er. 896 https://ww w.stats.ox.ac.uk/pu b/MA S S4/ . 897 Ve nces M, K osuch J, Boistel R, Ha d da d CF B, L a Marca E, L ötters S, Veit h M (2 00 3) Co nver ge nt 898 evoluti on of ap ose matic color ation i n N eot ropical p oiso n frogs: a m ol ecular p hylo ge netic 899 persp ective. Org anisms Diversity & Evolut ion, 3, 2 1 5– 22 6. 900 http://ww w.ur ba nfischer.d e/jo urn als/od e 901 Warre n R, V a nd erw al J, Price J, Welber g en JA, Atkins on I, R amirez-Ville g as J, Os bor n TJ, Jarvis A, 902 Sh oo LP, Willia ms SE, Lo we J (2 01 3) Qu antifying t he be nefit of e arly climate ch an g e mitig ation 903 in avoi din g bi odiversity loss. Nature Clima te Ch a ng e, 3(7), 6 7 8– 68 2. 904 https://doi.or g/1 0.1 03 8/nclim ate 18 8 7 905 Watters JL, C ummi ngs ST, Fl an a ga n R L, Siler C D (20 1 6) Revie w of m or ph om etric me asure me nts 906 used i n a nur an sp ecies d escriptions a n d recom me n datio ns for a sta nd ardize d a p proach. 907 Zo otaxa, 40 72( 4), 47 7 –4 9 5. https://doi.or g /10.1 1 64 6/zoot axa. 40 72. 4.6 908 909 910 911 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint Fig ure le ge n ds. 912 913 Fig ure 1. M a p of re se arc h ar ea. T h e sou thern half of th e P acific lowla nds of Col om bia, p art of the 914 Ch ocó bi og e ogr ap hical re gio n, h osts at least three sp ecies of Ep i ped ob a te s . Ph oto gra ph of the fro g 915 from Tim biq uí (R eserva R a nita terribilis, F un daci ón Pr oAv es de Col om bia) w as o bta ine d from 916 iNatur alist (observatio n N o. 1 35 2 53 84 3 by Juan Carlos Lu na); ot her im ag es w ere ta ken by th e 917 aut hors. 918 919 Fig ure 2. Im ag e s in life a nd in pre ser va tive of th e h olot yp e of E pip e do bat es [t o b e rev e ale d] 920 sp. n o v. . A F ull specim en in life; B Dors al view in life; C V entr al view in life; D Lat eral view in life; E 921 Dorsal vie w in pr eservative (7 0 % et h an ol); F Ve ntral view i n pres ervative; G L ateral view in 922 preservative; H Dorsal ha n d in pr eservative; I Ve ntral h an d in pr eservative; J Dorsal foot in 923 preservative; K Ve ntral fo ot in pr eservative. Scale bars re prese nt 5 m m (B to G) or 2.5 m m (H to K). 924 925 Fig ure 3. Im ag e s in life of Ep i pe dob a tes [ t o be r e vea l ed ] sp. no v. in c om pari s on wit h clo s e 926 co ng e ner s a nd sy mp atric s pe cie s. A. E . [to be reve ale d] sp. n ov. from the typ e lo cality of 927 La drilleros, V alle d e C auc a, C olom bia ( A ND ES: A:5 26 1; SV L = 2 0.0 mm; a dult m al e; par atype); B. E. 928 narin e nsis from Biot op o, Nari ño, Col om bi a (AN DE S:A: 37 0 4; 16. 39 mm; a dult m ale) ; C. An din ob ates 929 min utus from L a drilleros, Vall e d e C auc a, Col om bia (A ND ES: A:5 26 6; 1 3 m m; sex n ot det ermi ne d); D. 930 E. bo ule n geri from Isla G org o na, C a uca, Col om bia (in dividu al n ot capt ure d); E. E. espin osai fro m Río 931 Pal en qu e, S ant o D omi ng o d e los Tsác hila s, Ecuad or (individ u al not ca ptur ed); F. E. aff. espin osai 932 from L a N utria, Nari ño, C ol om bia (A ND ES :A:24 7 6; 17. 77 mm; sex n ot det ermi ne d); G. E. bo ule n geri 933 from M ara grícola, N ariñ o, Col om bia (A N D ES:A: 24 7 2; 18. 96 mm; a d ult fem ale); H. A side-by-side 934 ima ge of E. bo ul en geri from Isla G org o na (AND E S:A:3 6 95; 2 0.6 5 m m; ad ult fem ale) an d E. [to b e 935 reveal ed] sp. n ov. from Pi an g uita, V alle d e C auca, Col om bia (A ND E S:A:3 69 0; 1 6.4 2 mm; ad ult 936 femal e) d em onstrati ng th e lar ge size differ ence bet we e n the t wo sp ecies. All ima ges were t aken by 937 RD T exce pt for A a nd C, w hich w ere take n by JCR. P h otos are not to scal e. 938 939 Fig ure 4. Ph ylo ge n etic p ositio n a n d g e netic dist an ce s of E. [to be r ev eal ed] s p. no v. a nd oth er 940 sp eci es of E pip ed ob ate s . A A species-l evel m aximu m-likeliho o d p hylog eny of Epi pe do b ates 941 estimate d in IQ- TR EE usin g two nucle ar g en es ( BM P2, H 3 ) a nd t hre e mitoch o ndrial ge ne fra gm e nts 942 (C YT B , 12 S–1 6S , C R ) (se e Fi g. Su p p. m aterial S 2 for full p hylo ge ny). No de su p por t values from 943 10, 00 0 ultraf ast bo otstrap re plicates in dicate d strong s up port of E. [to be r eveal ed] sp. nov. as the 944 sister-group of E. n arin ensis . B Matrix of me an pairwis e p-dista nces a mo n g specie s of Epip ed o bat es . 945 Th e lo wer tria ngl e distanc es are base d o n CY TB a n d the up p er trian gle dista nces ar e from 12S –1 6S . 946 Epi pe do b ates [to b e reve ale d] is ge netical ly most similar to E. narin e nsis (1.77 % f or 12 S– 1 6S an d 947 5.3 9 % for CYT B ). A bbr eviatio ns: cur, E. [ to be r eveal e d] sp. nov; n ar, E. nari ne nsis ; bou N, E. 948 bo ule ng eri (Nari ño, Col om bia); b ou G, E. b oul en geri (Isla G org on a, Ca uca, Col om bia ); aesp, E. aff. 949 espin osai ; es p, E. espi nosai ; tri, E. tricolor ; mac, E. mach alilla ; a nt, E. a nth onyi . Se e Su pp. m aterial 950 S2 for full p hylo ge ny. 951 952 Fig ure 5. Co mp aris on s of S VL an d a d v ertise me nt c all traits bet we en E. [to b e rev eal ed] sp . 953 no v. a nd oth er sp e cie s of Ep i pe dob a tes . A S no ut-vent le ngt h (S VL) B Pe ak freq ue ncy C Call 954 dur ation an d D Nu mb er of p ulses p er call. Ab breviatio ns inclu de: E. bo u - Gor, E. b o ule ng eri - 955 Gorg o na; E. bo u - 956 Nar, E. b o ule ng eri - Nariñ o; E. n ar, E. n arine nsis; and E. cur sp. nov., E. [to b e reve ale d] sp. nov . 957 Black poi nts repres ent ra w d ata, th e h orizont al bar sh ows th e m ea n, th e sha de d di a gram is a 958 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint smoot he d d e nsity curve showin g th e full d ata distrib ution, an d th e recta ngl e repr ese nts the 959 uncert ainty aro u nd th e m ea n usi ng a 9 5 % Bayesia n hi gh est de nsity interval. 960 961 Fig ure 6 . Vo calizatio n s in E. [to b e re ve ale d] sp. no v. a nd oth er sp e cie s of Ep ip ed oba t es . 962 Sp ectrogr ams (a bove) an d oscillogr ams (bel ow) sh owi ng ge n eral asp ects of the structure of on e 963 spont an e ous a dvertisem e nt call for on e m ale of A E. [to b e reve ale d] sp. n ov. of La d rilleros, Valle del 964 Ca uca, B E. nari ne nsis of Bioto p o, Nari ño , C E. bo ule n geri -G org on a (Typ e loc ality) an d D E. 965 bo ule ng eri -N ariñ o of M ara grícola, N ariñ o. Advertise me nt calls consist of call grou pin gs, comprisin g a 966 single call for E pip e do bat es [to be r eve ale d] sp. nov., 2 –3 calls for E. b oul en g eri -Go rgo na, 3 – 6 calls 967 for E. bo ule n geri -N ariñ o, a nd 5– 14 calls for E. n arine nsis . E Sp ectrogr ams a n d oscillogra ms sho win g 968 ge ner al asp ects of the structure of on e co urtship call of E. [to b e rev eal ed] sp. n ov. of La drilleros, 969 Valle del Ca uca. F Co mp ariso n of o ne ad vertisment call b etw ee n E. [to b e reve ale d ] sp. nov. an d 970 oth er species of Ep i ped ob a te s . E. bo u - G or: E. bo ule n geri - Gor go n a, E. b ou - N ar: E. bo ule n geri - 971 Nariñ o, E. n ar: E. n arin ensis an d E. cur sp . nov.: E. [to be rev eal ed] sp. n ov . Fast F o urier Tra nsform-972 FF T = 2 56, ov erla p = 9 0 %. 973 974 Fig ure 7. Differen c es i n the ac o ustic pa ramet ers of E. [to b e rev eal ed] sp. n o v. co mp are d to 975 oth er sp e cie s of E pip ed o bat es . A Th e r elative co ntributi on of eac h aco ustic variable to th e first five 976 princip al com po ne nts obt ain e d in th e Prin cipal C om po ne nt A nalysis. The abs olut e valu e of e ach 977 contrib ution is re prese nte d accor din g to th e size of the circle, whil e blu e a n d red col ors show positive 978 an d n eg ative co ntributi ons, resp ectively. Call d urati on, n um b er of p ulses an d int ercall interval ar e 979 strongly a nd positively associate d with PC 1 w hile hi gh fre q ue ncy an d 9 0 % b a nd widt h are stro ngly 980 an d n eg atively associate d wit h PC 1. P C2 is strongly an d p ositively related to l ow fre qu ency a nd pe ak 981 frequ e ncy, while th e freq u ency inter qu artile b an dwi dth 90 % is n e gatively associate d with P C2. B A 982 Lin ear Discrimin ant An alysis using PCs a s input in dicates E. [to be rev eal ed] ca n b e differe ntiat ed 983 from oth er sp ecies bas ed on th eir a dvertisem ent calls, particul arly regar din g call dur ation an d n u mb er 984 of puls es (PC1 l oa ds p ositively on L D1). Dots indic ate th e in dividu als, color indicat es the sp ecies, 985 an d ellips es indicat e the 9 5 % co nfide nce i ntervals for L DA dat a p oints. 986 987 Fig ure 8: Ha bitat stru ctur e of Ep i pe doba t es [ t o be r e vea l ed ] sp. no v. at tw o loc alities. A– C 988 Imag es of th e type loc ality at La drilleros, Bu en ave ntur a, Vall e d el C auca, Col om bia . Usually, this 989 species is foun d o n ro adsi des n ear strea ms forme d by rai nfall. D– F At th e type l ocality, we o bserved 990 frogs in h abit ats conta min ate d with gar ba ge or agricult ural w aste. N ote th e frog i n F (red circle). G–J 991 Ha bitat char acteristics in Anchicayá, D ag ua, V alle del C a uca, C olo mbi a. Ima ges B, C an d E wer e 992 taken by RD T, all ot hers by M BC. 993 994 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 27, 2024. ; https://doi.org/10.1101/2024.03.23.586415doi: bioRxiv preprint