Mesoamerican tree squirrels evolution (Rodentia: Sciuridae): a molecular phylogenetic analysis Federico Villalobos1,2* & Gustavo Gutierrez-Espeleta3 1. Laboratorio de Genética Evolutiva, Escuela de Ciencias Biológicas, Universidad Nacional de Costa Rica, Heredia, Costa Rica. 2. Programa de Doctorado en Ciencias, Facultad de Ciencias, Universidad de Costa Rica, San José, Costa Rica; [email protected] 3. Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica; [email protected] * Correspondence Received 22-VII-2013. Corrected 13-I-2014. Accepted 23-I-2014. Abstract: The tribe Sciurini comprehends the genera Sciurus, Syntheosiurus, Microsciurus, Tamiasciurus and Rheinthrosciurus. The phylogenetic relationships within Sciurus have been only partially done, and the relation- ship between Mesoamerican species remains unsolved. The phylogenetic relationships of the Mesoamerican tree squirrels were examined using molecular data. Sequence data publicly available (12S, 16S, CYTB mitochondrial genes and IRBP nuclear gene) and cytochrome B gene sequences of four previously not sampled Mesoamerican Sciurus species were analyzed under a Bayesian multispecies coalescence model. Phylogenetic analysis of the multilocus data set showed the neotropical tree squirrels as a monophyletic clade. The genus Sciurus was para- phyletic due to the inclusion of Microsciurus species (M. alfari and M. flaviventer). The South American species S. aestuans and S. stramineus showed a sister taxa relationship. Single locus analysis based on the most compact and complete data set (i.e. CYTB gene sequences), supported the monophyly of the South American species and recovered a Mesoamerican clade including S. aureogaster, S. granatensis and S. variegatoides. These results corroborated previous findings based on cladistic analysis of cranial and post-cranial characters. Our data sup- port a close relationship between Mesoamerican Sciurus species and a sister relationship with South American species, and corroborates previous findings in relation to the polyphyly of Microsciurus and Syntheosciurus’ paraphyly. Rev. Biol. Trop. 62 (2): 649-657. Epub 2014 June 01. Key words: phylogenetics, cytochrome B, Sciurus, Microsciurus, Syntheosciurus, Mesoamerica. Tree squirrels are distributed throughout griseus and S. arizonensis). The genus reaches Eurasia and America, and grouped in the tribe its highest diversity in the Neotropical region Sciurini (Black, 1963; Wilson & Reeder, 2005). with 20 species. Sciurini comprehends the genera Sciurus, Syn- Among the Neotropical Sciurus species, S. theosiurus, Microsciurus, Tamiasciurus and variegatoides, S. yucatanensis and S. richmon- Rheinthrosciurus. Among these, the genus Sci- di are restricted to the Mesoamerican region urus, with seven subgenera and 28 species -the area between the Isthmus of Tehuantepec (Wilson & Reader, 2005), is the most diverse and the Isthmus of Panama-. Several other spe- and widespread, with representatives in Eur- cies (e.g. S. deppei, S. granatensis, and S. col- asia (S. vulgaris), the Japan Islands (S.lis), the liaei) occur in the Mesoamerican region albeit Middle East (S. anomalus) and the Americas. not exclusively. In addition to Sciurus, there In the Americas, 25 Sciurus species occur, are other Sciurini genera with Mesoamerican with several species restricted to the Neartic endemic taxa; the genus Microsciurus currently region (S. carolinensis, S. niger, S. aberti, S is composed by four species: M. alfari and M. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 62 (2): 649-657, June 2014 649 mimuls distributed in lower Central Ameri- the validity Synteosciurus, suggesting that S. ca, and M. flaviventer and M. santandarensis brochus should be considered a Sciurus spe- found in Northern South America. The genus cies, and also pointed to the possible poly- Syntheosciurus presents only one species with phyly of Microsciurus. two subspecies, S. brochus poaensis and S. The phylogenetic relationships within the brocus brocus, endemic of Talamanca-Chiriqui Sciurini tribe have been the focus of several mountain range in Costa Rica and Panama. studies in recent years. An early study by Haf- The most complete taxonomic revision of ner et al. (1994) based on protein variation the Sciurini was made by Moore (1959) based showed Microsciurus flaviventer in a Sciurus on the analysis of qualitative characters from clade comprising S. carolinensis, S. niger and the cranium. In this study, the genus Sciurus S. stramineus. The pioneering work of Mercer was placed in the subtribe Sciurina together & Roth (2003), based on the mitochondrial with Reinthrosciurus Gray and Guerlinguetus (12S rDNA and 16S rDNA) and nuclear genes Gray, which was later placed as a subgenus (IRBP), brought some information on the intra- in Sciurus (Hall, 1981). An additional sub- tribe relationships and showed the paraphy- tribe (Microsciurina) was also proposed for letic nature of the Sciurus genera in relation Microsciurus and Syntheosciurus. In relation to to Syntheosciurus and Microsciurus, and the Microsciurus, the genus was described initially relationship of these genera with Tamiasciurus. as a Sciurus´ subgenus (Allen, 1895) and later These same results were recovered by Steppan elevated to full generic rank (Goldman, 1912). et al. (2004) using two nuclear genes (RAG1 A similar situation occurred with Syntheosci- and cmy) and by Herron et al. (2004) using a urus, formerly considered a subgenus of Sci- mitochondrial gene (cytb). The close relation- urus (Goodwin, 1943, 1946). These affinities ship between M. flaviventer and the South- between Mesosciurus (currently S. granaten- american Sciurus species is a common feature sis) and the species in Microsciurus were also throughout these studies. Despite these relevant suggested by Moore (1959). results, the relationships within Sciurus and The phylogenetic relationships within Sci- its related genera remain largely unresolved. urus have been only partially resolved. Several The current data information include several studies have corroborated the monophyly of S. genes (nuclear and mitochondrial for a total vulgaris and S. lis (Oshida, Masuda, & Yoshi- of 8) but remains poor in the number of taxa da, 1996; Oshida & Masuda, 2000; Oshida, included and represented, precluding the pos- Arslan, & Noda, 2009). Also, previous analyses sibilities of a more comprehensive estimation based on an incomplete taxon sampling have of the phylogenetic relationships. A recent suggested that South American species might combined supermatrix approach using all gene form a monophyletic clade (Hafner, Barkley sequence publicly available (Pečnerová & Mar- & Chupasko, 1994; Steppan, Storz, & Hoff- tínková, 2012) also revealed the same relation- mann, 2004; Herron, Castoe, & Parkinson, ships between taxa. However, the phylogenetic 2004; Oshida et al., 2009). Moreover, the most relationships between Mesoamerican species species comprehensive phylogenetic study on remained unresolved, only supporting with low neotropical tree squirrels to date (Villalobos probability the sister relationship between M. & Cervantes, 2007) demonstrated the mono- alfari and S. granatensis. phyly of the Mesoamerican squirrel species. The methodological approach of concate- In that study, Mesoamerican species were nated supermatrix multilocus analysis has been split in two clades including S. aureogaster, S. recently point it as a possible source of error in colliaei, S. variegatoides and Syntheosciurus phylogenetic estimation. First revealed by Mad- brochus, and S. richomondi, S. granatensis, dison (1997), the phylogenetic trees depicted S. deppei, S. yucatanensis and Microsciurus by particular gene histories not necessarily alfari, respectively. These findings questioned correspond with the species tree phylogeny due 650 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 62 (2): 649-657, June 2014 to processes like horizontal transfer, lineage the supermatrix approach has shown a high sorting, gene duplication and gene extinction. probability of error estimating species trees, In conditions of short branches, closely related only 9 of 100 replicates contained the true spe- species and large population numbers the dis- cies tree topology (Heled & Drummond, 2010). crepancy between gene trees and species is In this study, we aim at resolving the phy- expected to be more problematic (Madisson, logenetic relationships of the Mesoamerican 1997; Maddison & Knowles, 2006). Therefore, tree squirrels using a Bayesian multispecies the use of several independent loci to estimate coalescence model. We provide new sequence the species tree was suggested (Pamilo & Nei, data (CYTB) of four Mesoamerican Sciurus 1998) and adopted as thought concatenation of species and used the newly generated informa- several genes, as a common practice in phylo- tion together with published DNA sequences. genetic reconstruction. However, recent studies into multigene phylogenetics have shown that MATERIALS AND METHODS concatenating genes can lead to poor estima- tion of species tree (Degnan & Rosenberg, Specimens analyzed: Twenty one taxa, 2006; Kubatko, 2007). When multiple loci are representing all recognized genera New World concatenate they are assumed to evolve accord- Tree squirrels, were included in the phylo- ing a single evolutionary tree, obviating the genetic analysis, (Table 1). The outgroup effect of recombination in decoupling the evo- taxa included Tamiasciurus hudsonicus, T. lutionary histories of different