1
0.03 L. parbocagii L. barbouri L. parkeri L. argenteus L. natalensis L. yaldeni L. vannutellii L. ragazzii L. susanae L. gramineus L. fiziensis L. vermiculatus L. kivuensis L. flavomaculatus L. bocagii L. modestus L. palmatus L. aubryioides L. notatus L. brevirostris L. calcaratus L. boulengeri L. ocellatus L. aubryi L. viridis L. spiritusnoctis L. macrotis L. millsoni L. rufus Astylosternus spp. (58mm) (43mm) (56mm) (52mm) (65mm) (43mm) (46mm) (50mm) (53mm) (63mm) (38mm) (85mm) (36mm) (70mm) (58mm) (41mm) (110mm) (43mm) (74mm) (64mm) (57mm) (81mm) (58mm) (54mm) (48mm) (49mm) (84mm) (87mm) (87mm) (NA) Size Morphological Analyses Among other characters, the extent of webbing in feet has been a consistently distinguishable tool for our specimens. We have also found that the ratio of disc (DSC) to Tympanum (TMP) and TMP to head width (HDW) or head length (HDL) is useful in our samples as well. We continue to explore new features that may be incorporated into the literature for future aid in identification. Deciphering Evolutionary Ancestry in African Tree Frogs: Genus Leptopelis Kyle E. Jaynes 1,2 and Rayna C. Bell 1 1 Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution 2 Department of Biology and Environmental Sciences, Adrian College West and Central African Leptopelis species (L. rufus, L. macrotis, and L. millsoni) were previously thought to be the closest ancestors of L. palmatus due to both their large size and proximity to the island chain. According to our most recent data, along with more extensive sampling from our collaborators, we find that L. palmatus is not closely related to any of the species mentioned above. Our results indicate that L. palmatus is within a group of Central African species that are smaller in body size, suggesting that there may be a convergence in gigantism within this genus. Moving forward, we are going to (1) increase our species and genetic sampling to determine which species of Leptopelis L. palmatus is closely related to, and (2) collect ddRADseq data for the L. macrotis/ L. millsoni/ L. rufus complex and look at more specimens to assess the morphological variation in these populations. These data will provide greater insight into the biogeography of land bridge and oceanic islands in the Gulf of Guinea archipelago and diversification in the Guineo-Congolian forests. In addition, we hope to find additional morphological characteristics to improve field identifications in this challenging species complex. Conclusion We collected 16s mitochondrial gene sequences for 33 species of Leptopelis and 146 individuals for our four target species. To determine whether L. palmatus is closely related to the L. rufus/ L. millsoni/ L. macrotis complex we generated a phylogeny using BEAST v 1.8.4 including one representative sequence for each species, with Astylosternus as an out-group. We assessed phylogeographic structure across the L. rufus/ L. millsoni/ L. macrotis complex with more than 100 specimens from 19 localities amongst the three species. We took 14 standard morphological measurements on more than 30 specimens to evaluate field identifications and search for more reliable morphological characters for species identification. Methods In figure 1 (a), Leptopelis palmatus is clearly separated from the L. macrotis/ L. millsoni/ L. rufus complex with strong support (>95% CI). There is not strong support for anything beyond the divergence of these groups, although it appears that there is a closer relationship with species of the Guinean-Congolian distribution. In figure 2 (a), we see clear phylogeographic structure across the species complex with support for distinct lineages of L. macrotis in West Africa and L. millsoni in Central Africa, distinct lineages of L. millsoni on Bioko island, and pronounced phylogeographic structure in L. rufus across Cameroon. Results Frost, Darrel R. 2017. Amphibian Species of the World: an Online Reference. Version 6.0 (07.24.2017). Electronic Database accessible at http://research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York, USA. Jones, P.J. 1994. Biodiversity in the Gulf of Guinea: an overview. Biodiversity and Conservation. 3:772-784. Linder, H.P., K.M. de Klerk, J. Born, N.D. Burgess, J. Fjeldsa, and C. Rahbek. 2012. Journal of Biogeography. 10:1365-2699. Schiotz A. 1999. Treefrogs of Africa. Chimaira. 1:251-258. References We would like to thank our collaborators on the project: Adam Leaché, David Blackburn, Eli Greenbaum, Dan Portik, Greg Jongsma, Matt Fujita, and Mark Oliver-Rödel. We thank Gracia Gonzalez Porter and the L.A.B. for assistance with sequences and facilities. We thank the NHRE program coordinators at the NMNH, Smithsonian Institution for their contributions to the REU program. This research was funded by NSF OCE: 1560088 and an ADS research grant. Acknowledgments Photo credit: A. Stanbridge Leptopelis palmatus Leptopelis palmatus Photo credit: A. Stanbridge The genus Leptopelis is a group of African Tree Frogs that have a poorly understood taxonomic history, with 53 currently accepted species (Frost 2017). These species are closely related and difficult to distinguish by morphological features alone, often times being misidentified in the field. Leptopelis palmatus is an endemic species to Príncipe Island, one of four islands in the Gulf of Guinea archipelago (Jones 1994). Reconstructing its colonization history and subsequent speciation is key to understanding the biogeography of this archipelago. L. macrotis, L. millsoni, and L. rufus form a species complex that occurs across West and Central Africa, as well as on Bioko Island, a continental land-bridge system. These species have been hypothesized to be the closest ancestors of L. palmatus due to their large size and proximity to Príncipe (Schiotz 1999). In addition, their distribution across continental Africa and Bioko Island make them an ideal study system for investigating speciation across a range of environments. In this project, we investigate questions of phylogeography and ancestral history in order to learn more about the evolutionary relationships amongst the organisms in this island system. Introduction Photo credit: A. Stanbridge Leptopelis palmatus Figure 1 (a) Phylogenetic tree of 16s mitochondrial gene. Includes 30 Leptopelis species with geographic distribution by color and maximum female size. Closed circles at nodes represent (>95% CI). Female size and distribution from (Schiotz et al 1999). (b) Biogeographical map of African ecosystems partitioned into 8 areas; Partitioned areas from (Linder et al. 2012). Biogeographical Regions of Africa Phylogeny of 30 Leptopelis Species 0.02 L_rufus_Cam_SWR_BYU_62290 L_rufus_Cam_SWR_BYU_62285 L_rufus_Cam_LR_CAS256878 L_rufus_Cam_LR_CAS254044 L_rufus_Cam_LR_CAS256881 L_rufus_Cam_LR_CAS_253837 L_macro_Guin_H_PB_11_642 L_macro_Lib_H_PLI_12_380 L_rufus_Cam_LR_CAS253968 L_macro_Lib_H_PGL_13_045 L_rufus_Cam_LR_CAS_254049 L_macro_Guin_D_MH0038_16 L_rufus_Cam_SWR_BYU_62291 L_rufus_Cam_SWR_BYU_62293 L_mills_Cam_Sd_CAS_253582 L_macro_Ghan_A_ZMB_71318 L_rufus_Cam_LR_CAS256684 L_rufus_Cam_LR_CAS256823 L_rufus_Cam_LR_CAS_256883 L_macro_Guin_H_PB_11_383 L_rufus_Cam_LR_CAS_254043 L_cf_rufus_Cam_EF_MH_0422 L_macro_Ghan_WR_UWBM8890 L_rufus_Cam_LR_CAS254050 L_mills_Cam_ER_CAS_253386 L_rufus_Cam_CR_CAS249976 L_macro_Guin_Z_PB_11_742 L_rufus_Cam_LR_CAS256882 L_rufus_Gabon_MCZ_139664 L_mills_Cam_Sd_CAS_253580 L_rufus_Cam_LR_CAS256879 L_macro_Lib_H_PLI_12_372 L_macro_Guin_H_PB_11_384 L_macro_Lib_H_PLI_12_373 L_rufus_Cam_LR_CAS256880 L_rufus_Cam_LR_CAS256877 L_rufus_Cam_LR_CAS256718 L_rufus_Cam_SD_CAS254099 L_rufus_Bioko_NCSM_84365 L_macro_Guin_H_PB_11_085 L_rufus_Nig_CR_MVZ_253291 L_rufus_Cam_SWR_BYU_62289 L_rufus_Cam_Sd_CAS_254098 L_rufus_Gabon_NCSM_77701 L_rufus_Cam_SWR_BYU_62288 L_rufus_Bioko_NCSM_84364 L_rufus_Gabon_MCZ_139665 L_mills_Cam_Sd_CAS_253581 L_rufus_Cam_SD_CAS254132 L_rufus_Cam_LR_CAS256680 L_macro_Liber_GNF_GRE_40 L_rufus_Cam_LR_CAS_253836 L_rufus_Nig_CR_MVZ_253292 L_rufus_Gabon_NCSM_76830 L_macro_Lib_H_PLI_12_218 L_rufus_Cam_LR_CAS_256876 L_rufus_Cam_LR_CAS256807 L_rufus_Cam_LR_CAS254046 L_rufus_Cam_LR_CAS254048 L_rufus_Gabon_MCZ_139662 L_rufus_Cam_LR_CAS254051 L_rufus_Bioko_NCSM_84366 L_rufus_Nig_CR_MVZ_253290 L_rufus_Cam_SD_CAS254133 L_cf_rufus_Cam_EF_MH_0215 L_mills_Cam_SWR_MCZ_136851 L_rufus_Cam_LR_CAS256808 L_rufus_Gabon_NCSM_77702 L_rufus_Cam_LR_CAS_253838 L_cf_rufus_Cam_EF_MH_0421 L_macro_Lib_V_PLI_12_186 L_rufus_Cam_LR_CAS256681 L_rufus_Cam_LR_CAS256717 L_rufus_Cam_SD_CAS254100 L_macro_Gui_FZ_CB2010_18 L_rufus_Cam_LR_CAS254045 L_rufus_Cam_SD_CAS254131 L_rufus_Gabon_MCZ_139713 L_rufus_Cam_BYU_62296 L_rufus_Cam_CR_CAS249982 L_rufus_Cam_CR_CAS249983 L_rufus_Cam_LR_CAS253839 L_macro_Guin_H_PB_11_282 L_rufus_Cam_LR_CAS256683 L_rufus_Cam_LR_CAS254052 L_rufus_Cam_CR_CAS249981 L_rufus_Cam_SWR_BYU_62286 L_rufus_Cam_LR_BYU_62298 L_mills_Cam_ER_CAS255080 L_rufus_Cam_LR_CAS254047 L_rufus_Cam_CR_CAS249984 L_rufus_Cam_LR_CAS256682 L_rufus_Cam_LR_CAS256806 L_rufus_Gabon_MCZ_139814 L_cf_rufus_Cam_MV_MH_0264 L_rufus_Cam_LR_CAS254042 L_cf_rufus_Cam_EF_MH_0242 L. rufus Cameroon - Littoral Region Cameroon Bioko Gabon Ghana Liberia- Guinea Cameroon - East Region L. millsoni L. macrotis Phylogeographic Tree of West-Central African Leptopelis Species Complex Leptopelis millsoni Photo credit: Rayna Bell Variation in Leptopelis Species Complex Photo credit: Rayna Bell Leptopelis palmatus Photo credit: B. Freiermuth Leptopelis rufus Leptopelis rufus Leptopelis millsoni Figure 3Hand sketch of webbing extent in L. millsoni and L. rufus. a) a) Sampling Map of West-Central African Leptopelis Species Complex b) b) L. Rufus Type Locality L. Millsoni Type Locality L. Macrotis Type Localtiy Figure 2 – (a) Phylogeographic tree of the L. rufus (red)/ L. macrotis (blue)/ L. millsoni (green) species complex, with geographically sampled and coded lineages. (b) Distribution map of L.macrotis (blue), L.millsoni (green), and L. rufus (red). Circle size is determined by the number of specimens sampled at that location. Triangles represent the type locality of that species. Background Photo Credit: http://wallpapercave.com/rain-forest-background Figures 1 and 2 B map outline credit: https://www.pinterest.com/pin/394627986069379882/

Deciphering Evolutionary Ancestry in African Tree Frogs ... · and Rayna C. Bell. 1 1. Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution

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Page 1: Deciphering Evolutionary Ancestry in African Tree Frogs ... · and Rayna C. Bell. 1 1. Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution

!!!!!!!!!!!!

0.03

L_spiritus

L_natalens

L_ocellatu

L_susanae

L_palmatus

L_flavomac

L_viridis

L_aubriodi

L_rufus

L_modestus

L_cf_ragaz

L_kivuensi

L_argenteu

L_vermicul

L_fiziensi

L_macrotis

L_vannutel

L_notatus

L_aubryi

L_yaldeni

L_gramineu

L_breviros

L_boulenge

Astylo_spp

L_parbocag

L_barbouri

L_bocagii

L_calcarat

L_parkeri

L_millsoni

!!!!!!!!!!!!!

L. parbocagii L. barbouri L. parkeri L. argenteus L. natalensis L. yaldeni L. vannutellii L. ragazzii L. susanae L. gramineus L. fiziensis L. vermiculatus L. kivuensis L. flavomaculatus L. bocagii L. modestus L. palmatus! L. aubryioides L. notatus L. brevirostris L. calcaratus L. boulengeri L. ocellatus L. aubryi L. viridis L. spiritusnoctis L. macrotis! L. millsoni! L. rufus! Astylosternus spp.

(58mm)! (43mm)! (56mm)! (52mm)! (65mm)! (43mm)! (46mm)! (50mm)! (53mm)! (63mm)! (38mm)! (85mm)! (36mm)! (70mm)! (58mm)! (41mm)! (110mm)! (43mm)! (74mm)! (64mm)! (57mm)! (81mm)! (58mm)! (54mm)! (48mm)! (49mm)! (84mm)! (87mm)! (87mm)! (NA)

Size

Morphological Analyses

Among other characters, the extent of webbing in feet has been a consistently distinguishable tool for our specimens. We have also found that the ratio of disc (DSC) to Tympanum (TMP) and TMP to head width (HDW) or head length (HDL) is useful in our samples as well. We continue to explore new features that may be incorporated into the literature for future aid in identification.

Deciphering Evolutionary Ancestry in African Tree Frogs: Genus Leptopelis Kyle E. Jaynes1,2 and Rayna C. Bell1

1Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution 2Department of Biology and Environmental Sciences, Adrian College

West and Central African Leptopelis species (L. rufus, L. macrotis, and L. millsoni) were previously thought to be the closest ancestors of L. palmatus due to both their large size and proximity to the island chain. According to our most recent data, along with more extensive sampling from our collaborators, we find that L. palmatus is not closely related to any of the species mentioned above. Our results indicate that L. palmatus is within a group of Central African species that are smaller in body size, suggesting that there may be a convergence in gigantism within this genus.

Moving forward, we are going to (1) increase our species and genetic sampling to determine which species of Leptopelis L. palmatus is closely related to, and (2) collect ddRADseq data for the L. macrotis/ L. millsoni/ L. rufus complex and look at more specimens to assess the morphological variation in these populations. These data will provide greater insight into the biogeography of land bridge and oceanic islands in the Gulf of Guinea archipelago and diversification in the Guineo-Congolian forests. In addition, we hope to find additional morphological characteristics to improve field identifications in this challenging species complex.

Conclusion

We collected 16s mitochondrial gene sequences for 33 species of Leptopelis and 146 individuals for our four target species. To determine whether L. palmatus is closely related to the L. rufus/ L. millsoni/ L. macrotis complex we generated a phylogeny using BEAST v 1.8.4 including one representative sequence for each species, with Astylosternus as an out-group. We assessed phylogeographic structure across the L. rufus/ L. millsoni/ L. macrotis complex with more than 100 specimens from 19 localities amongst the three species. We took 14 standard morphological measurements on more than 30 specimens to evaluate field identifications and search for more reliable morphological characters for species identification.

Methods

Results In figure 1 (a), Leptopelis palmatus is clearly separated from the L. macrotis/ L. millsoni/ L. rufus complex with strong

support (>95% CI). There is not strong support for anything beyond the divergence of these groups, although it appears that there is a closer relationship with species of the Guinean-Congolian distribution.

In figure 2 (a), we see clear phylogeographic structure across the species complex with support for distinct lineages of L. macrotis in West Africa and L. millsoni in Central Africa, distinct lineages of L. millsoni on Bioko island, and pronounced phylogeographic structure in L. rufus across Cameroon.

Results

Frost, Darrel R. 2017. Amphibian Species of the World: an Online Reference. Version 6.0 (07.24.2017). Electronic Database accessible at http://research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York, USA. Jones, P.J. 1994. Biodiversity in the Gulf of Guinea: an overview. Biodiversity and Conservation. 3:772-784. Linder, H.P., K.M. de Klerk, J. Born, N.D. Burgess, J. Fjeldsa, and C. Rahbek. 2012. Journal of Biogeography. 10:1365-2699. Schiotz A. 1999. Treefrogs of Africa. Chimaira. 1:251-258.

References

We would like to thank our collaborators on the project: Adam Leaché, David Blackburn, Eli Greenbaum, Dan Portik, Greg Jongsma, Matt Fujita, and Mark Oliver-Rödel. We thank Gracia Gonzalez Porter and the L.A.B. for assistance with sequences and facilities. We thank the NHRE program coordinators at the NMNH, Smithsonian Institution for their contributions to the REU program. This research was funded by NSF OCE: 1560088 and an ADS research grant.

Acknowledgments

Photo credit: A. Stanbridge Leptopelis palmatus

Leptopelis palmatus

Photo credit: A. Stanbridge

Introduction

The genus Leptopelis is a group of African Tree Frogs that have a poorly understood taxonomic history, with 53 currently accepted species (Frost 2017). These species are closely related and difficult to distinguish by morphological features alone, often times being misidentified in the field. Leptopelis palmatus is an endemic species to Príncipe Island, one of four islands in the Gulf of Guinea archipelago (Jones 1994). Reconstructing its colonization history and subsequent speciation is key to understanding the biogeography of this archipelago.

L. macrotis, L. millsoni, and L. rufus form a species complex that occurs across West and Central Africa, as well as on Bioko Island, a continental land-bridge system. These species have been hypothesized to be the closest ancestors of L. palmatus due to their large size and proximity to Príncipe (Schiotz 1999). In addition, their distribution across continental Africa and Bioko Island make them an ideal study system for investigating speciation across a range of environments. In this project, we investigate questions of phylogeography and ancestral history in order to learn more about the evolutionary relationships amongst the organisms in this island system.

Introduction

Photo credit: A. Stanbridge Leptopelis palmatus

Figure 1 – (a) Phylogenetic tree of 16s mitochondrial gene. Includes 30 Leptopelis species with geographic distribution by color and maximum female size. Closed circles at nodes represent (>95% CI). Female size and distribution from (Schiotz et al 1999). (b) Biogeographical map of African ecosystems partitioned into 8 areas; Partitioned areas from (Linder et al. 2012).

Biogeographical Regions of Africa Phylogeny of 30 Leptopelis Species

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!!!!

L. rufus! !!!

!!! Cameroon -

Littoral Region !!!!

Cameroon !! Bioko

Gabon

! Ghana

!! Liberia- Guinea !

! Cameroon - East Region !

!!!

L. millsoni! !!!!!!!!!!!!!!!!!!!

L. macrotis! !!!!!!!!

Phylogeographic Tree of West-Central African Leptopelis Species Complex

Leptopelis millsoni Photo credit: Rayna Bell

Variation in Leptopelis Species Complex

Photo credit: Rayna Bell Leptopelis palmatus

Photo credit: B. Freiermuth Leptopelis rufus

Leptopelis rufus Leptopelis millsoni

Figure 3– Hand sketch of webbing extent in L. millsoni and L. rufus.

a)

a)

Sampling Map of West-Central African Leptopelis Species Complex

b)

b)

L. Rufus Type Locality

L. Millsoni Type Locality

L. Macrotis Type Localtiy

Figure 2 – (a) Phylogeographic tree of the L. rufus (red)/ L. macrotis (blue)/ L. millsoni (green) species complex, with geographically sampled and coded lineages. (b) Distribution map of L.macrotis (blue), L.millsoni (green), and L. rufus (red). Circle size is determined by the number of specimens sampled at that location. Triangles represent the type locality of that species.

Background Photo Credit: http://wallpapercave.com/rain-forest-background Figures 1 and 2 B map outline credit: https://www.pinterest.com/pin/394627986069379882/