Schultz Et Al 2007 Lentulidae.Pdf

Systematic Entomology (2007), DOI: 10.1111/j.1365-3113.2007.00395.x Molecular phylogeny of the endemic East African flightless grasshoppers Altiusambilla Jago, Usambilla (Sjo¨ stedt) and Rhainopomma Jago (Orthoptera: Acridoidea: Lentulidae) OLIVER SCHULTZ1,2, CLAUDIA HEMP3 , ANDREAS HEMP4 and WOLFGANG WA¨GELE2 1Department of Animal Morphology & Systematics, University of Bochum, Germany, 2Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Germany, 3Department of Animal Ecology II, University of Bayreuth, Germany, 4Ecological Botanical Gardens, University of Bayreuth, Germany Abstract. A molecular phylogeny of endemic flightless grasshoppers is presented for the three Lentulidae genera Altiusambilla Jago, 1981, Usambilla Sjo¨stedt, 1909 and Rhainopomma Jago, 1981 based on DNA sequences (16S rRNA locus). Parsimony, distance and likelihood reconstructions were performed using different assumptions on sequence evolution. The generated phylogenies agree in almost all parts of the calculated trees and support the monophyly of the observed genera. It was shown that Usambilla and Rhainopomma are more closely related to each other, Altiusambilla being a separate clade. However, the investigated East African lentulid genera are clearly separated from South African taxa, underlining the monophyly of East African genera. Usambilla olivacea is re-established. Popula- tions of Rhainopomma montanum from the Taita Hills of Kenya and from the West Usambara mountains of Tanzania are two separate species not closely related to each other. Rhainopomma samples from the North Pare mountains of Tanzania belong to a hitherto undescribed species. Introduction due to the westward winds that constantly bring moisture from the Indian Ocean (Hamilton, 1989). The increased The family Lentulidae is a comparatively small family, with biodiversity and an accumulation of endemic species in this the two subfamilies Lentulinae (80 species in 21 genera) and region are comparable with Madagascar (Po´cs, 1998). Some the purely South African distributed subfamily Shelforditinae authors even described this area as the ‘Gala´pagos of Africa’ with mostly monotypic genera (ten genera and 13 species). (Rodgers & Homewood, 1982; Kingdon, 1990). In contrast Species of the investigated genera (Fig. 1) Altiusambilla to the old mountain formations of the Eastern Arcs Jago, 1981, Rhainopomma Jago, 1981 and Usambilla (estimated age approximately 30 My; Burgess et al., 1998), (Sjo¨stedt, 1909) mostly occur on currently climatically the volcanoes Kilimanjaro (1–3 My), Mount Meru (2 My) isolated high mountains or mountain ranges, such as the and Mount Kenya (5 My) are much younger (Marek, 2001). so-called Eastern Arc mountains (e.g. Pare mountains, To illuminate the speciation processes of flightless East Usambara mountains, Taita Hills) and inland volcanoes African Saltatoria, species of the genera Usambilla, Alti- (Fig. 2). The Eastern Arcs were ranked among the ten most usambilla and Rhainopomma were investigated. Usambilla important hotspots for endemism by Myers et al. (2000). and allies contain 33% of all known Lentulinae species. These mountain ranges harbour huge evergreen rainforests Their morphology and distribution patterns show that these species are closely related and in a recent radiation. Approximately half the 21 Lentulinae genera are restricted Correspondence: O. Schultz, Zoologisches Forschungsmuseum to southern Africa, few occur in tropical South Africa. Alexander Koenig, Adenauerallee 160, D-53113 Bonn, Germany. Almost 70% of all species of the family Lentulidae only E-mail: [email protected]. occur in southern Africa, with their diversity centre in the Unpublished for the purposes of zoological nomenclature (Art. 8.2. Cape Province of South Africa. Mecostibus is one of the ICZN). most species-rich genera of Lentulidae, with 13 species, and # 2007 The Authors Journal compilation # 2007 The Royal Entomological Society 1 2 O. Schultz et al. Fig. 1. Males and females of Rhainopomma, Usambilla and Altiusambilla.A,Rhainopomma usambaricum, male (East Usambara mountains, Tanzania); B, Usambilla emaliensis, male (Machakos, Kenya); C, Alitusambilla modicicrus, male (Mount Kilimanjaro, Tanzania); D, Rhainopomma usambaricum, female; E, Usambilla sp. (Mount Hanang, Tanzania), female; F, Altiusambilla modicicrus, female. shows the widest distribution in tropical South, North-east, genitalic morphology. Chromousambilla was based on West-central and East Africa. Genera investigated in this Adolfia latestriata Ramme and four additional species were paper have their main distribution in tropical East Africa. described by Jago (1981). This genus only occurs in north- Jago (1981) revised the genus Usambilla and its allies. He west Zambia and southern Tanzania. The monotypic erected the four new genera Chromousambilla, Microusam- Microusambilla is restricted to Zimbabwe and was erected billa, Altiusambilla and Rhainopomma on the basis of male for U. cylindricollis Ramme. The monotypic genus Altiusambilla Fig. 2. Map of East Africa in the area of the Eastern Arcs and inland volcanoes of Kenya and Tanzania. # 2007 The Authors Journal compilation # 2007 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/j.1365-3113.2007.00395.x Molecular phylogeny of East African Lentulidae 3 Table 1. Species of the genus Rhainopomma Jago. Original description Jago 1981 Distribution Adolfia usambarica Ramme R. usambaricum (Ramme) Tanzania, East Usambara; Kenya, Shimba Hills Usambilla montana Kevan R. montanum (Kevan) Kenya, Taita Hills; Tanzania, West Usambara, South Pare R. magnificum Jago Tanzania, South Pare R. nguruense Jago Tanzania, Nguru Mts. R. wapugu Jago Tanzania, Pugu Hills was erected for Lentula modicicrus Karsch from Mount entomological collections of the National Museums of Kilimanjaro. The type species of Rhainopomma is Adolfia Kenya, Nairobi, and the Naturkunde Museum, Berlin. usambarica Ramme from the East Usambara mountains of Tanzania. Jago (1981) placed U. montana Kevan into DNA extraction, amplification, purification and sequencing Rhainopomma and described three new species (Table 1). Jago (1981) transferred species from Lentula and Adolfia For all specimens listed in Table 3, muscle tissue of the into Usambilla, erected on U. olivacea Sjo¨stedt from the hind femur was used. Genomic DNA was extracted using West Usambara mountains of Tanzania, and described five different procedures: The QIAampÒ DNA mini kit (Qiagen, additional species and two new subspecies (Table 2). Most Germany), following the standard protocol for blood and species occur in East Africa. tissue, and the NucleoSpinÒ tissue kit (Machery & Nagel, He noted that, based on genitalic morphology, Altiusam- Germany) following the standard protocol for human and billa has affinities to Rhainopomma and especially to animal tissue. Mecostibus, but also stated that morphological similarities The extracted genomic DNA was used as a template for between Rhainopomma and Altiusambilla may be due to polymerase chain reaction (PCR) amplification with insect convergent evolution. primers modified for Saltatoria (16a: 59-CGC CTG TTT We analysed the phylogeny of East African species to ATC AAA AAC AT-39 and 16b: 59-CCG GTC TGA ACT understand the evolution of regional endemism and to CAG ATC ACG T-39; Kocher et al., 1989). Amplification determine the fit of these results with the existing taxonomy. was performed under the following conditions: initial denaturation 5 min at 94 8C; 38 cycles of 45 s at 94 8C, 45 s at 52 8C, 80 s at 72 8C, and a final extension step at Materials and methods 72 8C for 7 min. Amplification product (2 mL) was loaded on to 1.5% agarose Samples gels for size fractionation in gel electrophoresis to detect the amplification results (Sambook et al., 1989). A purification of Lentulid specimens were collected in the area of the East amplification products was performed using the standard African countries Kenya and Tanzania (Table 3, Fig. 2). protocol of the QIAquick PCR purification kit (Qiagen). Fresh specimens were fixed in 70% alcohol, then preserved Sequencing of the purified PCR products was conducted in several changes of 96% ethanol and stored at 4 8C. Air- in-house on a Licor DNA 4200 IR2 automated sequencer dried specimens were transferred into 96% ethanol and also using the thermo sequenase fluorescent labelled primer cycle stored at 4 8C. sequencing kit with 7-deaza-dGTP (Amersham, Germany) For each specimen, the locality, collection dates, altitude and on an ABI Prism 377 sequencer (Applied Biosystems, and habitat were noted. Table 3 lists the taxa for which U.S.A.) using the BigDye Ready Mix (Applied BioSystems). partial sequences of the mitochondrial 16s rRNA gene were Additionally sequencing was partly outsourced (Sequencing determined. Service, Macrogen, Korea). Additional sequences of Agathemera crassa (Blanchard, 1851) and the lentulid species Karruia sp., Lentula obtusi- frons1 Sta˚l, 1878 and Eremidium nr. equuleus (Karsch, 1896) Sequence analysis and phylogenetic reconstruction (selected from the NCBI GenBank) were chosen to root the phylogenetic trees. Sequence data were edited using the program SEQMAN 4.03 (DNAStar ). A basic local alignment search tool (BLAST; Identification. Lentulidae species were identified using Altschul et al., 1990) search at the NCBI GenBank the keys of Jago (1981). The material was checked again (www.ncbi.nlm.nih.gov) served to detect potential contam- against holotypes or paratypes of the Natural History ination. The alignment

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