ZooKeys 16A6 : n5e9w–7 3s p(2e0ci1e2s) of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomA epener-treovieswaed. .o.pen-access jo5urn9al doi: 10.3897/zookeys.166.2387 ReseARCH ARtiCle www.zookeys.org Launched to accelerate biodiversity research A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa (Poaceae, Bambusoideae) from Costa Rica Nicolás Pérez Hidalgo1,†, David Martínez-Torres2,‡, Jorge Mariano Collantes-Alegre2,§, William Villalobos Muller3,|, Juan M. Nieto Nafría1,¶ 1 Departamento de Biodiversidad y Gestión Ambiental, Universidad de León, E-24071, León, Spain 2 Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, E-46071, Valencia, Spain 3 Centro de Investigación en Biología Celular y Molecular, Universidad de Costa Rica, 11501-2060, San José, Costa Rica † urn:lsid:zoobank.org:author:B40B43A8-D339-499A-9370-C9D200D9F94C ‡ urn:lsid:zoobank.org:author:75388FFF-B033-4864-9CA5-455DE4FD9E15 § urn:lsid:zoobank.org:author:C2DA0012-1F1B-4A88-8082-20FFC13DD034 | urn:lsid:zoobank.org:author:FE514A95-E56A-4B81-9E57-BCA5504F530C ¶ urn:lsid:zoobank.org:author:81F0702F-538F-4AB4-92A9-74580A562221 Corresponding author: Nicolás Pérez Hidalgo ([email protected]) Academic editor: R. Blackman  |  Received 18 November 2011  |  Accepted 13 January 2012  |  Published 20 January 2012 urn:lsid:zoobank.org:pub:A898677B-81F5-49C5-9380-20A699FF571B Citation: Pérez Hidalgo N, Martínez-Torres D, Collantes-Alegre JM, Villalobos Muller W, Nieto Nafría JM (2012) A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa (Poaceae, Bambusoideae) from Costa Rica. ZooKeys 166: 59–73. doi: 10.3897/zookeys.166.2387 Abstract The new species Rhopalosiphum chusqueae Pérez Hidalgo & Villalobos Muller, is described from apterous viviparous females caught on Chusquea tomentosa in Cerro de la Muerte (Costa Rica). The identity of the species is supported both by the morphological features and by a molecular phylogenetic analysis based on a fragment of the mitochondrial DNA containing the 5’ region of the cytochrome c oxidase 1 (COI) and on the nuclear gene coding for the Elongation factor-1 alpha (EF1α). The taxonomic position of the new species is discussed. An identification key to the Aphidinae species living on plants of Bambusoideae (Poaceae) is presented. Keywords Rhopalosiphum, aphids, new species, molecular, Costa Rica Copyright Nicolás Pérez Hidalgo et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 60 Nicolás Pérez Hidalgo et al. / ZooKeys 166: 59–73 (2012) introduction The high diversity of organisms in Costa Rica has been referred to as a product of diverse ecosystems resulting from the interaction between complex microclimates, soils, topography, and a variety of biological processes, as well as the position of the country in the land-bridge between North and South America. Costa Rica’s biodi- versity comprises more than 500,000 species of organisms, approximately 84% of which are yet to be described. This percentage is even higher (90%) if we take insects, fungi, bacteria and viruses into account (Sánchez-Azofeifa et al. 2001). As for the number of aphid species present in Costa Rica, the list was recently extended (Pérez Hidalgo et al. 2009; Zamora Mejías et al. 2010; Villalobos Muller et al. 2010) and research is ongoing. During an expedition in 2008 in the area of Cerro de la Muerte (Cordillera de Talamanca), Costa Rica, three apterous viviparous females and several nymphs were collected on Chusquea tomentosa (Fig. 1). At first, they were assigned to the subtribe Rhopalosiphina Mordvilko, 1914 (Aphidini Latreille, 1802). This identifi- cation was confirmed in the laboratory when it was verified that the marginal papil- lae on abdominal segments I and VIII were in dorsal position to the corresponding stigmata. The morphological characters of the specimens resembled those of the genus Rhopalosiphum Koch, 1854, though the length of the setae were reminis- cent of species in the subgenus Paraschizaphis Hille Ris Lambers, 1947 (Schizaphis Börner, 1931). According to Valenzuela et al. (2009), Rhopalosiphum and Schizaphis form a mono- phyletic group with Melanaphis van der Goot, 1917, the separation between them be- ing unclear. The vein structure of the wings separates Schizaphis from Rhopalosiphum, however, as there were no alates available, molecular analyses were carried out to verify the relationship with the genus Rhopalosiphum through qualitative and quantitative characters. Molecular analyses are normally used to determine species and resolve taxo- nomic problems in the family Aphididae (Lozzier et al. 2008; Foottit et al. 2008, 2009; Lee et al. 2010; Pike et al. 2010). A microscopic morphotaxonomic study of the specimens enabled the hypothesis that they could not be assigned to any known species, and strengthened the hypothesis that they be assigned to Rhopalosiphum. Molecular phylogenetic analysis based (1) on a fragment of the mitochondrial DNA containing the 5’ region of the cytochrome c oxidase 1 (COI) and (2) on the nuclear gene coding for the Elongation factor-1 alpha (EF1α) were used to verify both hypotheses. Several species included in the subfamily Aphidinae are known living on plant species of the subfamily Bambusoideae (Poaceae), but only two belong to Rhopalosi- phum (Aphidini): Rhopalosiphum arundinariae (Tissot, 1933) and R. rufiabdominale (Schrank, 1899), and none to the genus Schizaphis. A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa... 61 Figure 1. A View of the area where the types of Rhopalosipum chusqueae were captured, in Cerro de la Muerte (Costa Rica), with Chusquea tomentosa B view of the plant C and D details of the area where the aphids were located. 62 Nicolás Pérez Hidalgo et al. / ZooKeys 166: 59–73 (2012) Material and methods Material studied Three apterous viviparous female and several nymphs (sample CRI-235) were recorded on Chusquea tomentosa Y. Widmer et L. G. Clark (Poaceae: Bambusoideae: Bambuse- ae: Chusquinae) in Ojo de Agua (Cerro de la Muerte, Cordillera de Talamanca, Costa Rica) (9°36'N, 83°47'W), 2968 m, 26.ii.2008. Morphological study Thirty-three quantitative characteristics and the qualitative features of shape, scleroti- zation, pigmentation and cuticular ornamentation, were considered. The method used for measurements is that normally employed in our studies (Nieto Nafría and Mier Durante 1998). A camera lucida fitted to the microscope was used for the drawings and the microphotographs were taken with a Leica DC digital camera with IM 1000 version 1.10 software. DNA extraction and PCR amplification Total DNA was extracted separately from two samples, one of them containing a single nymph and the second the contents of the abdomen of 3 apterous adults, all kept in 96% ethanol. We followed the HotSHOT (Hot Sodium Hydroxide and Tris) method (Truett et al. 2000). PCR amplification of the two gene fragments analyzed was carried out on 3 µl of the extracted DNA. A 710 bp fragment of the 5’ region of the mitochondrial cytochrome c oxidase subunit 1 (COI) was amplified using primers LCO1490 and HCO2198, described by Folmer et al. (1994). PCR conditions for COI amplifica- tion were as follows: 94°C for 1 min; 35 cycles of 94°C for 30 s, 48°C for 1 min and 68°C for 1 min; a final extension step of 7 min at 68°C was included after cycling. Amplification of the Elongation factor-1 alpha (EF1α) gene fragment was performed using two consecutive PCR reactions with primers Efs175 (Moran et al. 1999) and Efr1 (5’GTGTGGCAATSCAANACNGGAGT3’) in the first reaction and then primers Efs175 and Efr2 (5’TTGGAAATTTGACCNGGGTGRTT3’) in the second hemi-nested reaction. PCR conditions used in the first reaction were: 94°C for 1 min; 40 cycles of 94°C for 30 s, 50°C for 1 min and 68°C for 1.5 min; a final extension step of 7 min at 68°C was included after cycling. The hemi-nested PCR was done similarly but using 52°C for the annealing step and using 1 µl of the first PCR product. A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa... 63 Sequencing and analysis of DNA sequences PCR products were purified by ammonium precipitation and reconstituted in 10 µL of LTE buffer (10mM Tris, 0,1mM EDTA). Direct sequencing of amplified fragments was done in both directions using PCR primers (Efr2 was used as reverse primer for sequencing the EF1α fragment). Sequencing was conducted using the Big Dye Termi- nator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer’s instructions, and samples were loaded onto an ABI 3700 automated sequencer. Chromatograms were revised and sequences corresponding to each sample assem- bled using the Staden package v1.6.0 (Staden et al. 2000). Multiple alignments were carried out with Clustal X v1.81 (Thompson et al. 2002) with gap opening and gap extension penalties of 10.0 and 0.2, respectively, and subsequently manually revised. Phylogenetic analysis of COI sequences were done using MEGA version 4 (Ta- mura et al. 2007). For EF1α sequences ModelTest (Posada and Crandall 1998) was used to find the evolutionary model that best fitted sequence data and phylogenetic reconstruction was done using RAxML (Stamatakis et al. 2008). Results Morphological data A study of the qualitative and quantitative (metric and meristic) characters of the speci- mens enabled us to establish the hypothesis that they belong to the genus Rhopalosi- phum as, apart from the above-mentioned character of the marginal papillae on the abdomen, (1) when alive they are ovoid and when preserved the body is not very long and the margins are curved (Figs 2A, 2B), (2) the dorsal cuticle of the thorax and ab- domen is membranous, except for the presence of intersegmental sclerites and a pair of large sclerites on abdominal segment VIII (Figs 2A, 2B), (3) the dorsal cuticle has a more or less regular reticulate area formed by coalescent spinules (Figs 2Ba, 2Bb, 2F, 2G), (4) the siphunculi are longer than the cauda and clearly constricted underneath the apical edge (Figs 2A, 2B, 2D), and (5) there are few setae on the cauda. A comparison of the characters of these specimens with those of apterae in other species of Rhopalosiphum and Schizaphis also strengthened the hypothesis that they could not be assigned to any known species. Molecular data A 710 bp DNA fragment containing a portion of the mitochondrial COI gene was amplified through PCR from the two samples analysed. Useful sequences obtained 64 Nicolás Pérez Hidalgo et al. / ZooKeys 166: 59–73 (2012) Figure 2. Rhopalosiphum chusqueae sp. n. A, B Habitus C antennal segment III D siphunculus e cauda. F (and B-a) detail of the cuticule of abdominal segment 3 G (and B-b) detail of cuticule of abdominal segment 8. from each sample consisted of 658 nucleotides. Identical sequences were obtained for both samples so that a single sequence was finally assigned and deposited in Genbank (accession number HE604204). The online identification engine available at the Bar- code of Life Data Systems (BOLD) (Ratnasingham and Hebert 2007) using the COI A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa... 65 A B Figure 3. A Neighbour joining tree based on Kimura 2P distances obtained for the COI sequences from our new species (Rhopalosiphum chusqueae) and different Aphidini representatives obtained from the NCBI database. B Maximum Likelihood tree obtained for the EF1α sequences for our new spe- cies (Rhopalosiphum chusqueae) and different Aphidini representatives obtained from the NCBI database. Bootstrap support values obtained after 1000 replicates in A and 100 in B are indicated above branches if higher than 50%. Initials for genera are as follows: A, Aphis; As, Asiphonaphis; B, Braggia; H, Hyalopterus, M, Melanaphis; P, Paradoxaphis; R, Rhopalosiphum; S, Schizaphis (Schizaphis); S. (E.), Schizaphis (Euschi- zaphis); S. (P.), Schizaphis (Paraschizaphis); T, Toxoptera. 66 Nicolás Pérez Hidalgo et al. / ZooKeys 166: 59–73 (2012) species database, failed to find any record corresponding to any identified species that matched our sequence. After a BLASTN search against the non-redundant nucleotide database at the NCBI, sequences from different Rhopalosiphum species were most simi- lar to our sequence (93–94% identical) followed by Schizaphis sequences (92–93% identical). We then aligned our sequence with sequences from all Rhopalosiphum spe- cies available at the NCBI database and from species representative of closely related genera (Schizaphis, Melanaphis, etc.) that we had previously retrieved from the data- base, and built a phylogenetic tree (Fig. 3A). The tree shows that the sequence from our unknown species groups with relatively high support within a monophyletic clade that contains all other Rhopalosiphum and Schizaphis COI sequences occupying a rather basal position within that clade. For the Elongation factor-1 alpha (EF1α) gene fragment, we obtained an identi- cal sequence from the two analyzed samples of 987 bp which was deposited in the Genbank with accession number HE604205. Using sequences available for EF1α in NCBI for different Rhopalosiphum and closely related species within Aphidini, an ML tree was built that included the sequence obtained for our unknown species (Fig. 3B). As with the COI sequence, our unknown species grouped with strong support within a monophyletic clade that also included sequences from Schizaphis and Euschizaphis. However, unlike the COI tree, both Rhopalosiphum and Schizaphis-related sequences separated into two distinct clades, though with very low bootstrap support. Discussion and conclusion Molecular data using both mitochondrial COI and nuclear EF1α gene sequences con- firmed that the Rhopalosiphum chusqueae specimens belong to the same monophyletic clade as other Rhopalosiphum species occupying a rather basal position in the group likely closely related to other divergent Rhopalosiphum species such as R. nymphaeae. Both trees revealed the close relationship between Rhopalosiphum and Schizaphis gen- era. Although COI sequences are widely used in taxonomy, their utility for phylogeny reconstructions seems rather limited as their phylogenetic signal is somewhat weak in comparison with other markers (Wilson 2010). Contrarily, EF1α is widely used in phylogenetic reconstructions and its use in insect phylogeny has been shown to be in- formative (Simon et al. 2009; Wilson 2010). In this respect, although the COI analysis did not recover the monophyly of Rhopalosiphum and Schizaphis genera separately, ML analysis of EF1α separated both genera and clearly Rhopalosiphum chusqueae grouped within the Rhopalosiphum clade, which, along with our morphometric data discussed above, supports its assignation to the Rhopalosiphum genus. Approximately 15 species are classified in the genus Rhopalosiphum (Remaudière and Remaudière 1997; Zhang and Qiao 1997; Eastop and Blackman 2005; Black- man and Eastop 2006) associated with arboreal Rosaceae (Prunus or Pyroidea) as the primary host and with Poaceae, Cyperaceae or, less frequently, other plants as the sec- A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa... 67 ondary host if their cycle is dioecious, or only with one of them if their cycle is mo- noecious. Most of the species probably originate in North America, with a subsidiary centre of dispersal in Central Asia (Blackman and Eastop 1994; Halbert and Voegtlin 1998; Blackman and Eastop 2006). Five of its species have an exclusively Nearctic dis- tribution: R. arundinariae (Tissot), R. cerasifoliae (Fitch), R. enigmae Hottes & Frison, R. laconae Taber, R. nigrum Richards, and R. padiformis Richards; and another four Nearctic species have been introduced in other parts of the world: R. parvae Hottes & Frison and R. rufulum Richards in Europe, R. musae Schouteden has been recorded in areas of Europe, Central Asia, Africa and Australia, and R. oxyacanthae (Schrank) is known in Central- and South-America, Europe, Asia and Australia. To date, only four species, linked mainly to crops, have been recorded in Central American countries: R. maidis (throughout Central America), R. nymphaeae in Panama, R. padi in Costa Rica and Panama, and R. rufiabdominale in Honduras, Costa Rica and Panama (Evans and Halbert 2007; Quirós et al. 2009; Villalobos Muller et al. 2010); R. oxyacanthae is also known in Central America, without country (Blackman and Eastop 2006). Species of Rhopalosiphum most resembling the new species due to their mor- phological characters are R. rufiabdominale and R. padiformis. The former originated from East Asia (Blackman and Eastop 2006) and is currently widely distributed. R. padiformis originates from North America. R. chusqueae sp. nov. coincides with both species in the length of the setae, with R. padiformis in the number of antennal seg- ments and shape of the cauda, and with specimens of R. rufiabdominale in the 4 setae on abdominal segment VIII (R. rufiabdominale has 3 to 8 setae on this segment). It is easily distinguished from them because the antennae in R. rufiabdominale are five- segmented and the dorsal setae in R. padiformis are not pointed and abdominal seg- ment VIII only has 2 setae. Bamboo species are the host plants of the mentioned R. rufiabdominale and R. arundinariae; this last species can be easily differentiated from R. chusqueae by the shape of the cauda (short and more or less triangular or rounded) and siphunculus (more or less tapering) and by much shorter setae on body dorsum and appendages. In view of the above, a new species can be established, the description of which follows. Rhopalosiphum chusqueae Pérez Hidalgo & Villalobos Muller, sp. n. urn:lsid:zoobank.org:act:D3A0466B-3858-46A8-A4BB-4618BDD956D3 http://species-id.net/wiki/Rhopalosiphum_chusqueae Holotype. Apterous viviparous female number 1 of measurement series, caught on Chusquea tomentosa, Pérez Hidalgo & Villalobos Muller leg., deposited in the Aphido- logical Collection of the University of León (CZULE), sample CRI-235. Paratypes. 2 apterous viviparous females (in separated slides) caught with the holotype. 68 Nicolás Pérez Hidalgo et al. / ZooKeys 166: 59–73 (2012) Etymology. The specific epithet, chusqueae is the genitive singular of the generic name of the aphid’s host plant. Apterous viviparous females (Figure 2). When alive globular oval and brown with white spots of wax on abdomen. Mounted 2.20–2.72 mm and pale in general with head, antennae, legs, siphunculi and cauda dark-brown. Antennae 0.63–0.79 times body length. Antennal segment III (0.32–0.43 mm) shorter than segment IV (0.21–0.25 mm) plus V (0.20–0.26 mm); with setae 55–65 µm long and 1.8–2.6 times the articular diameter of the segment. Terminal process of segment VI (0.44–0.47 mm) 3.9–4.4 times the base (0.32–0.43 mm). Rostrum 0.52–0.61 mm long, reaching middle coxae, 0.19–0.27 times the body length. Ul- timate rostral segment 0.13–0.15 mm long, approximately 1.7 times its basal width and 1.1 second segment of hind tarsus; it carries two accessory setae. Marginal papillae present on prothorax, on the abdominal segment 1 and 7, which are dorsally placed to the respective spiracles, and sometimes on segments 3 and 6. Dorsum of the abdomen with spinules forming reticulate ornamentation. Dorsal setae on abdominal segment 3 with delicate, pointed and 25–30 µm long and 3.0–3.9 times the articular diameter of antennal segment III and shorter than ventral ones, which are 90–110 µm long. Siphunculi slightly swollen with marked narrowing below the flange, 0.41–0.45 mm long, 0.16–0.20 times the body length and 2.1–2.2 times cauda. Abdominal segment 8 with two sclerites and four setae 90–110 µm long, delicate and pointed. Genital plate with 2 discal setae and near 26 posterior ones. Cauda finger-like, 0.19–0.21 mm long and carrying 5 setae. Distribution and host-plant. Chusquea tomentosa (Poaceae, Bambusoideae) is the only known host of R. chusqueae. This bamboo is endemic to the country and can be found in several areas of the Cordillera de Talamanca at an altitude of between 2450 and 3000 m (Widmer 1997; Hammel et al. 2003). Species of Chusquea (approxi- mately 120 described) can be found at between 800 and 3800 m in dry and humid forests from Mexico to Chile and Argentina (Clark 1989). As species in the genus Rho- palosiphum are not strictly stenophagous, R. chusqueae may also live on other species of Chusquea, or even on other bamboos and live in other parts of America. On the plant, the aphids live close to the nodes well protected by the leaves (Figs 1C, 1D) and not easily detectable, as shown by fruitless efforts to locate other colonies. So far, only one aphid species had been recorded on Chusquea: Hysteroneura se- tariae (Thomas) on Chusquea abietifolia Griseb, in Cuba (Holman 1974). Blackman and Eastop (1994) present two identification keys to the aphid species living on Arundinaria and on Bambusa, genera that include arboreal bamboos; sev- eral of these aphid species belong to the subfamily Aphidinae. Blackman and Eastop (2006) report the presence of aphid species on several genera of non-arboreal bamboos such as Chusquea, Pseudosasa, Sinoarundinaria, Thamnocalamus, Thysanolaena, and also Arundinaria (other bamboo genera are included but no Aphidinae species have been recorded on them), and for the identification of these species the reader is forwarded to the “keys [of 1994] to aphids on Arundinaria and Bambusa”, or to the “keys [of